- Email: [email protected]
5. Development of Enhancing Intraosseous Delivery Efficiency of LV-Factor VIII Variants in Platelets of Hemophilia A Mice
RNA Virus Vectors support of this, LDLR surface expression and receptor density is unchanged following β-deliverin treatment. In β-deliverin treated cells, 2 hours post-transduction, we observed reduced accumulation of virus in late endosomes, a 3-fold reduction in the total number of late endosomes, as measured by LAMP1 puncta, and a nearly 2-fold increase in the proportion of early endosomes when compared to control cells. Together, our data imply that β-deliverin facilitates more efficient exit of virus from the endosome, possibly by inhibiting endosomal maturation. Further mechanistic studies are ongoing. Notably, a monomer of β-deliverin has no effect on HSC transduction, suggesting an important structure-function relationship. β-deliverin is a promising tool not only for the development of novel gene delivery applications, but also to further elucidate LVHSC interactions. We are investigating whether the mechanisms we observed here hold true for other small molecule transduction enhancers, and using these drugs to probe the biology of LV restriction in HSCs.
5. Development of Enhancing Intraosseous Delivery Efficiency of LV-Factor VIII Variants in Platelets of Hemophilia A Mice
Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses. These results indicated that F8X10K12 may further enhance FVIII gene expression in platelets for more effective therapy. LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated and were intraosseously delivered into HemA mice to test the FVIII efficiency in platelets by ELISA, thrombin generation assay and tail clipping. Conclusion: We found that administration of Dex that efficiently inhibited initial innate immune responses to LVs in vivo combined with anti-CD8α mAb that depleted subsequent cytotoxic CD8+ T cells improved the transduction efficiency of LVs and persistence of transduced cells, leading to over 10% GFP+HSCs in treated mice up to 160 days. In addition, a new FVIII variant, F8X10K12, can significantly enhance FVIII expression in mice following hydrodynamic injection of plasmids and in LV-transduced cells. Taken together, IO infusion of G-F8X10K12-LV into HemA mice pretreated with Dex and antiCD8α mAb can be used to further enhance and prolong transgene efficiency in platelets for effective correction of hemophilia A.
Xuefeng Wang, Richard Y. Fu, Carol H. Miao Seattle Children’s Research Institute, Seattle, WA
6. Retargeted Foamy Virus Vectors Integrate Less Frequently Near Proto-Oncogenes
Introduction: Our previous studies demonstrated that human factor VIII (FVIII) specifically expressed in megakaryocytes and then stored in platelets of Hemophilia A (HemA) mice can partially correct their phenotype over 5 months in mice with or without preexisting inhibitors. This was achieved by intraosseous (IO) delivery of lentiviral vectors (LVs) carrying a transgene encoding human FVIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) without preconditioning as required in ex vivo gene therapy. Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents. The other was to improve FVIII gene expression by incorporating a new human FVIII variant, F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain; a kind gift from Dr. Weidong Xiao). Results: First, the immune competent C57BL6 mice were pretreated with both dexamethasone (Dex) (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21), or Dex only. IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0. On day 7, Dex only and combination drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (17.7±3.5% and 11.8±2.1% vs 6.9±3.1%, P=0.0001 and 0.005) and GFP+Lineage-Sca1+cKit+ HSCs (55.5±3.1% and 48.3±6.1% vs 44.4±17.2%, P=0.1 and 0.31) compared with LV-only treated mice (n=3). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P<0.001) in the total bone marrow and GFP+HSCs (10.7±3.3% vs 2.6±0.6%, P<0.001) were observed in combination drugs + LVs treated mice (n=3) compared with LVonly treated mice on day 160 after LV infusion (n=3), which was confirmed by higher LV copy number in bone marrow cells of drugs + LVs treated mice. Second, we tested the FVIII expression levels from two human FVIII variants in HemA mice by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1αF8X10K12 or pEF1α-F8, 50 μg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Then two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively.
Hematopoietic stem cell gene therapy offers immense potential to treat many genetic diseases and has already shown efficacy in clinical trials. However, retroviral vector mediated gene dysregulation, known as genotoxicity, remains a major challenge and clinically relevant approaches to reduce integration near genes and proto-oncogenes are needed. The frequency at which a retroviral vector integrates near proto-oncogenes is a major factor in determining the safety of the vector. Here we describe a clinically relevant method to retarget foamy retroviral vector (FV) integration away from proto-oncogenes using modified Gag and Pol helper constructs. The chromatin binding site (CBS) of FV Gag was altered using a previously described triple alanine substitution mutation of RTY (Gag-RTY) shown to drastically reduce chromatin binding of the FV pre-integration complex. The modified FV Pol construct expresses chromobox protein homolog 1 fused to the C-terminus of integrase (CBX1-IN). CBX1 interacts with tri-methylated lysine 9 of histone H3 (H3K9me3) and is associated with gene sparse regions in the human genome. We hypothesized a modified FV expressing Gag-RTY and CBX1-IN would have an altered integration site profile and be retargeted to H3K9me3 regions and away from genes and proto-oncogenes. FV integration sites from modified and control FVs were compared in normal human fibroblasts and in CD34+ human cord blood cells. We observed retargeting of FV integration into H3K9me3 regions with the modified FV (Figure 1). Importantly, retargeting FV integration significantly reduces the number of integration sites near proto-oncogene transcription start sites (Table 1). Retargeted FVs can be produced at clinically relevant titers (> 107 transducing units / ml), resulting in efficient transgene expression (75 % of control in CD34+ cells) with no evidence of vector silencing. Another published method to retarget lentiviral vector integration required using a modified cell line, which is not practical for clinical use. Our approach is cell line independent and the modified Gag and Pol helper constructs will allow an investigator to simply use these modified helper plasmids during vector production to retarget any therapeutic FV in any target cell. Retargeted FVs integrate less frequently near proto-oncogenes than gammaretroviral vectors and unmodified FVs, and may be the safest current option for hematopoietic stem cell gene therapy.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Jonah D. Hocum, Ian Linde, Dustin T. Rae, Casey P. Collins, Lindsay K. Matern, Grant D. Trobridge Pharmaceutical Sciences, Washington State University, Spokane, WA
S3
5. Development of Enhancing Intraosseous Delivery Efficiency of LV-Factor VIII Variants in Platelets of Hemophilia A Mice
Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses. These results indicated that F8X10K12 may further enhance FVIII gene expression in platelets for more effective therapy. LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated and were intraosseously delivered into HemA mice to test the FVIII efficiency in platelets by ELISA, thrombin generation assay and tail clipping. Conclusion: We found that administration of Dex that efficiently inhibited initial innate immune responses to LVs in vivo combined with anti-CD8α mAb that depleted subsequent cytotoxic CD8+ T cells improved the transduction efficiency of LVs and persistence of transduced cells, leading to over 10% GFP+HSCs in treated mice up to 160 days. In addition, a new FVIII variant, F8X10K12, can significantly enhance FVIII expression in mice following hydrodynamic injection of plasmids and in LV-transduced cells. Taken together, IO infusion of G-F8X10K12-LV into HemA mice pretreated with Dex and antiCD8α mAb can be used to further enhance and prolong transgene efficiency in platelets for effective correction of hemophilia A.
Xuefeng Wang, Richard Y. Fu, Carol H. Miao Seattle Children’s Research Institute, Seattle, WA
6. Retargeted Foamy Virus Vectors Integrate Less Frequently Near Proto-Oncogenes
Introduction: Our previous studies demonstrated that human factor VIII (FVIII) specifically expressed in megakaryocytes and then stored in platelets of Hemophilia A (HemA) mice can partially correct their phenotype over 5 months in mice with or without preexisting inhibitors. This was achieved by intraosseous (IO) delivery of lentiviral vectors (LVs) carrying a transgene encoding human FVIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) without preconditioning as required in ex vivo gene therapy. Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents. The other was to improve FVIII gene expression by incorporating a new human FVIII variant, F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain; a kind gift from Dr. Weidong Xiao). Results: First, the immune competent C57BL6 mice were pretreated with both dexamethasone (Dex) (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21), or Dex only. IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0. On day 7, Dex only and combination drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (17.7±3.5% and 11.8±2.1% vs 6.9±3.1%, P=0.0001 and 0.005) and GFP+Lineage-Sca1+cKit+ HSCs (55.5±3.1% and 48.3±6.1% vs 44.4±17.2%, P=0.1 and 0.31) compared with LV-only treated mice (n=3). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P<0.001) in the total bone marrow and GFP+HSCs (10.7±3.3% vs 2.6±0.6%, P<0.001) were observed in combination drugs + LVs treated mice (n=3) compared with LVonly treated mice on day 160 after LV infusion (n=3), which was confirmed by higher LV copy number in bone marrow cells of drugs + LVs treated mice. Second, we tested the FVIII expression levels from two human FVIII variants in HemA mice by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1αF8X10K12 or pEF1α-F8, 50 μg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Then two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively.
Hematopoietic stem cell gene therapy offers immense potential to treat many genetic diseases and has already shown efficacy in clinical trials. However, retroviral vector mediated gene dysregulation, known as genotoxicity, remains a major challenge and clinically relevant approaches to reduce integration near genes and proto-oncogenes are needed. The frequency at which a retroviral vector integrates near proto-oncogenes is a major factor in determining the safety of the vector. Here we describe a clinically relevant method to retarget foamy retroviral vector (FV) integration away from proto-oncogenes using modified Gag and Pol helper constructs. The chromatin binding site (CBS) of FV Gag was altered using a previously described triple alanine substitution mutation of RTY (Gag-RTY) shown to drastically reduce chromatin binding of the FV pre-integration complex. The modified FV Pol construct expresses chromobox protein homolog 1 fused to the C-terminus of integrase (CBX1-IN). CBX1 interacts with tri-methylated lysine 9 of histone H3 (H3K9me3) and is associated with gene sparse regions in the human genome. We hypothesized a modified FV expressing Gag-RTY and CBX1-IN would have an altered integration site profile and be retargeted to H3K9me3 regions and away from genes and proto-oncogenes. FV integration sites from modified and control FVs were compared in normal human fibroblasts and in CD34+ human cord blood cells. We observed retargeting of FV integration into H3K9me3 regions with the modified FV (Figure 1). Importantly, retargeting FV integration significantly reduces the number of integration sites near proto-oncogene transcription start sites (Table 1). Retargeted FVs can be produced at clinically relevant titers (> 107 transducing units / ml), resulting in efficient transgene expression (75 % of control in CD34+ cells) with no evidence of vector silencing. Another published method to retarget lentiviral vector integration required using a modified cell line, which is not practical for clinical use. Our approach is cell line independent and the modified Gag and Pol helper constructs will allow an investigator to simply use these modified helper plasmids during vector production to retarget any therapeutic FV in any target cell. Retargeted FVs integrate less frequently near proto-oncogenes than gammaretroviral vectors and unmodified FVs, and may be the safest current option for hematopoietic stem cell gene therapy.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Jonah D. Hocum, Ian Linde, Dustin T. Rae, Casey P. Collins, Lindsay K. Matern, Grant D. Trobridge Pharmaceutical Sciences, Washington State University, Spokane, WA
S3