- Email: [email protected]
6. In Vivo Adult Stem Cell Gene Transfer in Mice by In Situ Delivery of a Self-Inactivating Lentiviral Vector Using Intrafemoral Injection
RNA VIRUS VECTORS: GENE TRANSFER AND GENE EXPRESSION hematopoiesis linear amplification mediated (LAM)-PCR was used for all animal samples studied. In all animals analyzed multiple clones could be detected and in animals that received both G-PBSC and G-BM multiple clones contributed from both transduced cell populations. These in vivo marking results were not in agreement with the report by Hematti et al so we sought to determine whether the different results might be explained by the fact that different combinations of cytokines (G-CSF and SCF) for mobilization modulate receptor expression of the amphotropic (PIT2) and GALV (PIT1). Analysis of receptor expression in baboon CD34+ cells after administration of different combinations of cytokines for mobilization found receptor expression was dramatically affected by different mobilization regimens with G-CSF and SCF increasing PIT2 expression and G-CSF alone increasing PIT1 expression. The low in vivo gene marking in previous reports using amphotropic pseudotyped vectors and G-CSF alone for mobilization could be explained because in animals treated with G-CSF alone, at least in baboons, the amphotropic receptor expression was minimal while including SCF in the mobilization regimen substantially increased amphotropic receptor expression. In contrast to other studies, our high in vivo marking in G-CSF-mobilized baboon peripheral blood CD34+ cells demonstrates the feasibility of efficiently transducing G-PBSC in clinical gene therapy trials and verifies that there is no inherent stem cell deficiency in G-PBSC mobilized cells.
6. In Vivo Adult Stem Cell Gene Transfer in Mice by In Situ Delivery of a Self-Inactivating Lentiviral Vector Using Intrafemoral Injection D. Nicole Worsham,1 Kimberly Bohn,1 David Kuhel,1 David A. Williams,1,2 Christof von Kalle,1,2 Dao Pan.1,2 1 Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Department of Pediatics, University of Cincinnati, Cincinnati, OH. The potential of in vivo bone marrow stem cell gene transfer by bone cavity injection has not been explored. This approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if efficient gene transfer can be achieved in HSC and mesenchymal stem cells (MSC) by in situ delivery of a HIV-based lent virus vector (LV), we intrafemorally injected into adult normal mice (Ly5.1) with a selfinactivating GFP-containing LV (at 2 x106 IU/mouse). GFPexpressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. The colony-forming cell (CFC) assay resulted in 4-5% of GFP-expressing CFUs with multilineage potentials 4-months after injection, which was consistent with 4color FACS analysis data demonstrating up to 3.7% of GFP+ HSC/ progenitors (Lin-c-kit+Sca1+). Higher frequencies of GFP-containing CFUs (up to 15.9%) were revealed by a colony-direct duplex PCR. To further evaluate HSC gene transfer, bone marrow from injected mice was transplanted into lethally irradiated adult C57BL/6 (Ly5.2) mice with successful engraftment demonstrated by 97.5-98.5% donor-derived PBL in all recipients 3-months post BMT. Four months after transplantation, we observed 8.4 to 17.7 % GFPexpressing CFUs, as well as 16 to 24% GFP-containing CFUs in all recipients. Vector integrants and their multi-clonality were further confirmed by linear-amplification mediated PCR (LAM-PCR) in both primary injected and secondary transplanted mice. We also demonstrated that GFP-expressing MSC retained multiple differentiation potential. Our data provide the first evidence that adult stem cells in bone marrow could be efficiently transduced in vivo by “in situ” vector administration. This may potentially open a door to a novel approach for treatment of human diseases. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
7. Transduction of Quiescent Human Cells Using an SIVsmmPBj-Derived Lentiviral Vector Matthias Schweizer,1 Nina Wolfrum,1 Michael Muehlebach,2 Julia Kaiser,1 Silke Schuele,1 Klaus Cichutek.1 1 Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany; 2Molecular Medicine Program, Mayo Clinic, Rochester, MN. Although lentiviral vectors enable gene transfer into nonproliferating cells, particular quiescent human cells remain resistant to lentiviral transduction. Actually, several potential target cells of human gene therapy, particularly cells in the G0-state of the cell cycle such as freshly isolated primary monocytes, cannot be transduced using conventional HIV- or SIV-derived vectors. In contrast to HIV and most SIV strains, SIVsmmPBj from sooty mangabey monkey is able to replicate in non-stimulated peripheral blood lymphocytes from primates, indicating that an SIVsmmPBjderived (PBj) vector may allow gene transfer into quiescent cells. Therefore, various PBj vectors pseudotyped with the VSV-G glycoprotein and transferring the egfp gene were generated and tested for their ability to transduce different target cells. PBj vectors, in contrast to corresponding HIV-1-derived vectors, were able to transduce G0-arrested human cell lines or diploid fibroblasts, independently from the presence of the accessory viral genes vif, vpr, vpx or nef. Furthermore, PBj vectors enabled transduction of primary human monocytes already during the first days after isolation, whereas HIV-1-derived vectors mediated transduction only after differentiation into macrophages or dendritic cells. Using Southern blot analysis and a two step Alu-PCR assay, chromosomal integration of the PBj vector DNA into the target cell genome was demonstrated. However, transduction of monocytes was not possible using PBj vectors lacking the viral accessory gene vpx. By complementation of these vectors with Vpx, the transduction capacity could be reconstituted, confirming the importance of Vpx for transduction of monocytes. In summary, SIVsmmPBj-derived vectors offer advantages over HIV-1-derived vectors in transduction of certain quiescent human cells.
8. Potent Inhibition of HIV-1 Replication by Lentiviral Vectors Carrying Anti-Vif siRNA S. Li,1 G. Wang,2 K. Meinking,1 M. J. Li,2 J. Ji,2 J. A. Zaia,1 J. J. Rossi.2 1 Virology, City of Hope Beckman Research Institute, Duarte, CA; 2 Molecular Biology, City of Hope Beckman Research Institute, Duarte, CA. Small inhibitory RNAs (siRNAs) inhibit HIV replication by promoting sequence-specific degradation of genomic and sub-genomic HIV-1 RNA. Lentiviral vectors are able to transduce a wide array of quiescent cell types with sustained long-term expression of the transgenes. RNA polymerase III (pol III) provides an ideal means of expressing small RNA in human cells. HIV-1 viral infectivity factor (Vif) is one of the six regulatory proteins encoded by HIV-1. It has been previously reported that Vif is accessible to RNAi in the incoming viral RNA. We therefore directed 21-nucleotide siRNA duplexes against three different regions of HIV-1 Vif sequences, and incorporated the siRNA genes into a lentiviral vector backbone under the control of the human U6 pol III promoter with green fluorescent protein (GFP) as a marker for transduction. We transduced the vectors into CEM cells, sorted the GFP positive cells, and challenged the cells with HIV-1NL4-3. To evaluate the anti-HIV activity we analyzed HIV genomic RNA, HIV cDNA and p24 antigen production. Since Vif is a late gene product, we re-challenged CEM cells with supernatants from the original infectious assays to monitor reduction in viral infectivity as a consequence of Vif knockdown.
S3
6. In Vivo Adult Stem Cell Gene Transfer in Mice by In Situ Delivery of a Self-Inactivating Lentiviral Vector Using Intrafemoral Injection D. Nicole Worsham,1 Kimberly Bohn,1 David Kuhel,1 David A. Williams,1,2 Christof von Kalle,1,2 Dao Pan.1,2 1 Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Department of Pediatics, University of Cincinnati, Cincinnati, OH. The potential of in vivo bone marrow stem cell gene transfer by bone cavity injection has not been explored. This approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if efficient gene transfer can be achieved in HSC and mesenchymal stem cells (MSC) by in situ delivery of a HIV-based lent virus vector (LV), we intrafemorally injected into adult normal mice (Ly5.1) with a selfinactivating GFP-containing LV (at 2 x106 IU/mouse). GFPexpressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. The colony-forming cell (CFC) assay resulted in 4-5% of GFP-expressing CFUs with multilineage potentials 4-months after injection, which was consistent with 4color FACS analysis data demonstrating up to 3.7% of GFP+ HSC/ progenitors (Lin-c-kit+Sca1+). Higher frequencies of GFP-containing CFUs (up to 15.9%) were revealed by a colony-direct duplex PCR. To further evaluate HSC gene transfer, bone marrow from injected mice was transplanted into lethally irradiated adult C57BL/6 (Ly5.2) mice with successful engraftment demonstrated by 97.5-98.5% donor-derived PBL in all recipients 3-months post BMT. Four months after transplantation, we observed 8.4 to 17.7 % GFPexpressing CFUs, as well as 16 to 24% GFP-containing CFUs in all recipients. Vector integrants and their multi-clonality were further confirmed by linear-amplification mediated PCR (LAM-PCR) in both primary injected and secondary transplanted mice. We also demonstrated that GFP-expressing MSC retained multiple differentiation potential. Our data provide the first evidence that adult stem cells in bone marrow could be efficiently transduced in vivo by “in situ” vector administration. This may potentially open a door to a novel approach for treatment of human diseases. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
7. Transduction of Quiescent Human Cells Using an SIVsmmPBj-Derived Lentiviral Vector Matthias Schweizer,1 Nina Wolfrum,1 Michael Muehlebach,2 Julia Kaiser,1 Silke Schuele,1 Klaus Cichutek.1 1 Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany; 2Molecular Medicine Program, Mayo Clinic, Rochester, MN. Although lentiviral vectors enable gene transfer into nonproliferating cells, particular quiescent human cells remain resistant to lentiviral transduction. Actually, several potential target cells of human gene therapy, particularly cells in the G0-state of the cell cycle such as freshly isolated primary monocytes, cannot be transduced using conventional HIV- or SIV-derived vectors. In contrast to HIV and most SIV strains, SIVsmmPBj from sooty mangabey monkey is able to replicate in non-stimulated peripheral blood lymphocytes from primates, indicating that an SIVsmmPBjderived (PBj) vector may allow gene transfer into quiescent cells. Therefore, various PBj vectors pseudotyped with the VSV-G glycoprotein and transferring the egfp gene were generated and tested for their ability to transduce different target cells. PBj vectors, in contrast to corresponding HIV-1-derived vectors, were able to transduce G0-arrested human cell lines or diploid fibroblasts, independently from the presence of the accessory viral genes vif, vpr, vpx or nef. Furthermore, PBj vectors enabled transduction of primary human monocytes already during the first days after isolation, whereas HIV-1-derived vectors mediated transduction only after differentiation into macrophages or dendritic cells. Using Southern blot analysis and a two step Alu-PCR assay, chromosomal integration of the PBj vector DNA into the target cell genome was demonstrated. However, transduction of monocytes was not possible using PBj vectors lacking the viral accessory gene vpx. By complementation of these vectors with Vpx, the transduction capacity could be reconstituted, confirming the importance of Vpx for transduction of monocytes. In summary, SIVsmmPBj-derived vectors offer advantages over HIV-1-derived vectors in transduction of certain quiescent human cells.
8. Potent Inhibition of HIV-1 Replication by Lentiviral Vectors Carrying Anti-Vif siRNA S. Li,1 G. Wang,2 K. Meinking,1 M. J. Li,2 J. Ji,2 J. A. Zaia,1 J. J. Rossi.2 1 Virology, City of Hope Beckman Research Institute, Duarte, CA; 2 Molecular Biology, City of Hope Beckman Research Institute, Duarte, CA. Small inhibitory RNAs (siRNAs) inhibit HIV replication by promoting sequence-specific degradation of genomic and sub-genomic HIV-1 RNA. Lentiviral vectors are able to transduce a wide array of quiescent cell types with sustained long-term expression of the transgenes. RNA polymerase III (pol III) provides an ideal means of expressing small RNA in human cells. HIV-1 viral infectivity factor (Vif) is one of the six regulatory proteins encoded by HIV-1. It has been previously reported that Vif is accessible to RNAi in the incoming viral RNA. We therefore directed 21-nucleotide siRNA duplexes against three different regions of HIV-1 Vif sequences, and incorporated the siRNA genes into a lentiviral vector backbone under the control of the human U6 pol III promoter with green fluorescent protein (GFP) as a marker for transduction. We transduced the vectors into CEM cells, sorted the GFP positive cells, and challenged the cells with HIV-1NL4-3. To evaluate the anti-HIV activity we analyzed HIV genomic RNA, HIV cDNA and p24 antigen production. Since Vif is a late gene product, we re-challenged CEM cells with supernatants from the original infectious assays to monitor reduction in viral infectivity as a consequence of Vif knockdown.
S3