Humanized High-Expression Factor VIII Encoding Lentiviral Vectors Efficiently Drive High-Level FVIII Expression in Murine and Human Hematopoietic Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3543-3543
Author(s):  
Christopher Doering ◽  
Gabriela Denning ◽  
Keith Kerstann ◽  
Bagirath Gangadharan ◽  
Robert Keefe ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Hematopoietic Cells ◽  
Lentiviral Vectors ◽  
High Expression ◽  
Human Cell Lines ◽  
Hek 293 ◽  
Fviii Activity ◽  
Cd34 Cells ◽  
High Level

Abstract Several hurdles limit the successful gene therapy treatment of hemophilia A. For example, human coagulation factor VIII (fVIII) is inefficiently biosynthesized and has proven difficult to express using recombinant viral gene transfer, target cells have been inefficiently transduced, and fVIII expression can result in inhibitory antibody formation to the transgene product. Recently, we showed that B-domain-deleted porcine fVIII (BDDpfVIII) is expressed at levels 100-fold higher than BDD human fVIII (BDDhfVIII) in both in vitro and in vivo systems. Ex vivo modification of murine bone marrow cells with recombinant BDDpfVIII-encoding retrovirus followed by transplantation into non-myeloablative hemophilia A murine recipients resulted in long-term fVIII expression at levels considered curative, and the transplanted mice were immunogenically tolerant to the BDDpfVIII expressed from the genetically-modified hematopoietic cells. We now have identified the residues within BDDpfVIII responsible for the high-level expression and incorporated them into the cDNA encoding BDDhfVIII, thus generating a high-expression hybrid human/porcine (HP) construct composed of 90% human and only 10% porcine sequence. High titer recombinant oncoretroviral and lentiviral vectors were generated with the chimeric HP-fVIII sequence and used to transduce human cell lines, murine sca-1+ cells, and primary human hematopoietic cells. The lentiviral vectors efficiently transduced the human cell lines HEK-293, Hela, K562, EU-1, jurkat and U937, the former 4 being hematopoietic cell-derived. In HEK-293 cells, a linear increase in fVIII expression was observed with increasing MOI, and at 7-days after transduction the expression was 28 units/106 cells/24 hr, compare to only 4 units/106 cells/24 hr for BDDhfVIII (MOI = 3). In the hematopoietic cells lines, therapeutically significant, but approximately 10-fold lower expression was observed. Transplantation of oncoretroviral or lentiviral transduced murine hematopoietic stem and progenitor cells into lethally conditioned hemophilia A recipient mice resulted in long-term fVIII expression at therapeutic levels (>10% normal human levels) despite having only 5% or less genetically-modified blood mononuclear cells. No fVIII activity was observed in hemophilia A mice transplanted under identical conditions using recombinant virus encoding BDDhfVIII. Human CD34+ cells were isolated from fresh bone marrow aspirates, cultured overnight, and then transduced with either lentivirus encoding eGFP or the chimeric HP-fVIII (MOI = 5). Similar numbers of progenitor colonies grew in methylcellulose cultures for each construct indicating no increased toxicity resulting from HP-fVIII expression. Real-time PCR using genomic DNA isolated from pooled colonies from methylcellulose plates showed gene-marking levels of approximately 0.3 proviral genomes/diploid genome equivalent, thus indicating a transduction efficiency of approximately 30%. Furthermore, fVIII activity levels were greater in CD34+ cell cultures transduced with vectors encoding chimeric HP-fVIII (0.1 units/106 cells/24 hr) than negative control CD34+ cells transduced with vectors encoding eGFP (<0.01 units106 cells/24 hr). From these studies, it is concluded that humanized high-expression HP-fVIII transgenes can be utilized to significantly increase fVIII expression levels in the context of future clinical gene transfer-based therapies for hemophilia A.

High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors

Blood ◽  
2001 ◽  
Vol 98 (9) ◽  
pp. 2664-2672 ◽  
Author(s):  
Francois Moreau-Gaudry ◽  
Ping Xia ◽  
Gang Jiang ◽  
Natalya P. Perelman ◽  
Gerhard Bauer ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Hepatitis Virus ◽  
Globin Gene ◽  
Hematopoietic Cells ◽  
Lentiviral Vectors ◽  
Specific Gene ◽  
High Level Expression ◽  
Specific Expression ◽  
Cd34 Cells ◽  
High Level

AbstractUse of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34+ cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/β-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34+ cells. Sca1+/lineage− Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40–containing vector encoding a hybrid human β/γ-globin gene led to 43% to 113% human γ-globin expression/copy of the mouse α-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.

FVIII Tolerance Following Nonmyeloablative Transplantation of Hematopoietic Stem Cells Engineered To Encode a High-Expression Factor VIII Transgene in Hemophilia A Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2599-2599
Author(s):  
Lucienne M. Ide ◽  
Bagirath Gangadharan ◽  
Christopher Doering ◽  
H. Trent Spencer
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Hemophilia A ◽  
Genetically Modified ◽  
High Expression ◽  
Activity Levels ◽  
Fviii Activity ◽  
Inhibitory Antibodies ◽  
High Level

Abstract Clinical gene therapy trials for hemophilia A have failed due to insufficient target cell transduction or expression of factor VIII (fVIII). Animal studies have documented the extreme difficulty of expressing therapeutic levels of human fVIII from recombinant viral vectors without eliciting an anti-fVIII humoral response. We previously demonstrated curative fVIII activity levels in hemophilia A mice following a nonmyeloablative, genetically-modified bone marrow transplantation (Ide et al., Blood2007). In these studies recipient hemophilia A mice received 3×105 bone marrow-derived sca-1+ cells transduced with a retrovirus encoding B-domain-deleted high-expression porcine fVIII (BDDpfVIII) after pre-transplantation conditioning with reduced-intensity TBI or busulfan followed by minimal immunosuppression with either costimulation blockade or antithymocyte serum (ATS). Using our high-expression fVIII transgene and busulfan or 3Gy TBI coupled with ATS resulted in sustained fVIII activity of &gt;100% normal human levels. The main hematopoietic toxicity observed was transient T-cell suppression. High-level fVIII activity (&gt;2 units/ml) was achieved without induction of anti-BDDpfVIII humoral immune responses. Furthermore because the development of inhibitory antibodies directed against fVIII remains a significant clinical complication associated with the treatment of hemophilia A, we tested the effectiveness of genetically-modified cells encoding BDDpfVIII in the setting of pre-existing fVIII immunity. Transplantation of genetically-modified HSCs into 5.5 Gy TBI/ATS or busulfan/ATS-conditioned hemophilia A mice that had significant anti-human fVIII inhibitory titers induced sustained, high-level fVIII activity. Following transplantation and stable engraftment, both naïve and pre-immunized mice were challenged with six weekly injections of 10 units human fVIII, an immunization regimen that results in antibody formation in 100% of naïve hemophilia A mice. All mice sustained their pre-challenge BDDpfVIII activity levels, and anti-fVIII antibodies were not detected. Surface markers of T-cell activation were the same between naïve hemophilia A mice and transplanted mice, for both naïve and preimmunized animals. In a mixed lymphocyte reaction, T-cells were found to be equally capable of reacting to allogenic antigens (i.e. irradiated Balb/C splenocytes) when isolated from mice with stable fVIII expression compared to nontransduced controls, demonstrating that the lack of anti-fVIII inhibitors is not due to a lack of immunocompetent T-cells. Further studies are underway assessing T-cell nonresponsiveness to BDDpfVIII in these transplanted mice. By demonstrating that transplanted mice are tolerant to BDDpfVIII, even in the context of pre-existing inhibitory antibodies and nonmyeloablative conditioning, this data further supports our previous studies showing that retroviral gene transfer using high-expression porcine fVIII elements is a compelling treatment for hemophilia A.

scholarly journals Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1297-1305 ◽  
Author(s):  
N Uchida ◽  
J Combs ◽  
S Chen ◽  
E Zanjani ◽  
R Hoffman ◽  
...  
Keyword(s):  
Stromal Cell ◽  
Biological Activities ◽  
Single Cells ◽  
Hematopoietic Cells ◽  
In Utero ◽  
Rhodamine 123 ◽  
Differentiation Potential ◽  
Cd34 Cells

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy- 1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy- 1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.

Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2587-2587
Author(s):  
Hideto Matsui ◽  
Margareth Ozelo ◽  
Carol Hegadorn ◽  
Andrea Labelle ◽  
Erin Burnett ◽  
...  
Keyword(s):  
Immune Response ◽  
Intravenous Injection ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Therapeutic Potential ◽  
Antigen Presenting Cells ◽  
Lentiviral Vectors ◽  
Target Sequence ◽  
Antigen Presenting

Abstract Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.

Long-Term Polyclonal and Stable Gene Transfer into Rhesus Repopulating Hematopoietic Stem Cells Using a Simian Immunodeficiency Virus-Based Lentiviral Vector System.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3259-3259
Author(s):  
Yoo-Jin Kim ◽  
Nadia L. Hussein ◽  
Peiman Hematti ◽  
Bum-Kee Hong ◽  
Boris Calmels ◽  
...  
Keyword(s):  
B Cells ◽  
Gene Transfer ◽  
Simian Immunodeficiency Virus ◽  
Vector System ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
High Level

Abstract Murine leukemia virus (MLV) vectors have been studied extensively in animal models and utilized for over a decade in clinical trials of gene therapy directed at hematopoietic stem and progenitor cells MLV have a number of limitations, including inefficient transduction of quiescent cells and difficulty in maintaining stable high-level expression. More recently concerns have arisen regarding their safety regarding activation of adjacent proto-oncogenes and resultant leukemogenesis. We have previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus CD34+ cells, resulting in high-level in vivo marking with transduced progeny cells up to one year post-transplantation.(Hanawa et al, 2004) A comparison of vector integration sites in these animals compared to animals receiving MLV-transduced cells revealed different patterns, showing that SIV integrants strongly favored entire transcription units and gene-dense regions of the genome, compared to MLV that favored regions surrounding transcription start sites.(Hematti et al, 2004). Animals receiving MLV-transduced cells had highly non-random engraftment with integrants in or near the the MDS1/EVI1 gene complex. To evaluate long-term safety implications of the SIV vector-mediated CD34+ cell gene transfer, we analyzed the insertional sites in granulocytes, T cell, and B cells from 3 rhesus macaques which were transplanted three years ago with transduced, autologous cytokine-mobilized peripheral blood CD34+ cells. All three animals continued to show significant marking and expression levels in T cells, B cells and granulocytes, with mean GFP + levels of 6.7% (range, 3.3–13.0%), 7.4% (4.2–13.4%) and 5.6% (3.1–10.5%), respectively. Vector insertion site analysis by linear amplification-mediated PCR method at three years continued to show highly polyclonal reconstitution. Subsequent cloning and sequencing data confirmed long-term polyclonality with vector-containing cells and there was no evidence for any worrisome common integration sites, with no integrants detected in the MDS1/EVI1 region, in contrast to results with the MLV vector. These results indicate that the SIV vector system can result in stable and efficient long-term expression in progeny of transduced CD34+ cells, without the worrisome integration profile previously reported in our model with MLV vectors.

Development of a New Generation, Forward-Oriented Therapeutic Vector for Hemoglobin Disorders

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1172-1172 ◽  
Author(s):  
Naoya Uchida ◽  
Matthew M. Hsieh ◽  
Aylin C Bonifacino ◽  
Allen E Krouse ◽  
Mark E Metzger ◽  
...  
Keyword(s):  
Globin Gene ◽  
Transduction Efficiency ◽  
Erythroid Cells ◽  
Cd34 Cells ◽  
Globin Promoter ◽  
High Level ◽  
Viral Components ◽  
Intron 2 ◽  
Viral Titers

Abstract Ameliorating hemoglobin disorders such as sickle cell disease (SCD) using hematopoietic stem cell (HSC) gene therapy is under development. Unlike in other diseases, therapeutic globin vectors have demanding requirements including high-level β-globin expression, tissue specificity among erythroid cells, long-term persistence, and high-level modification at the HSC level. These demanding requirements necessitate the inclusion of complex genetic elements including the locus control region (LCR), β-globin promoter, β-globin gene, and the 3' untranslated region (3'UTR), all now feasible using lentiviral vectors. The additional requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent loss by RNA splicing during viral preparation. This reverse-orientation is in contrast to all other therapeutic vectors under clinical development. Current reverse-oriented globin vectors can drive phenotypic correction in mouse models for both b-thalassemia and SCD, while they are limited by lower viral titers and lower transduction efficiency in primary human HSCs, limiting their prospects, especially in SCD. We hypothesized that the reverse-orientation impedes both viral preparation and vector transduction, as despite deletion of cryptic polyadenylation (polyA) signals to optimize a conventional reverse-oriented globin vector, titers were still 10-fold lower than a standard GFP-vector. We thus designed a forward-oriented globin-expressing vector, which was further optimized by minimizing the size of the LCR, inclusion of a large segment of the β-globin promoter and an enhancer region of the 3'UTR lacking the polyA signal. Viral titers of the forward-oriented vectors (1.0±0.2x10e9 IU/mL) were 6-fold higher than the optimized vector in the reverse orientation (1.6±0.2x10e8 IU/mL, p<0.01), and comparable to a standard GFP-marking vector (1.9±0.2x10e9 IU/mL, p<0.01). The forward-oriented vector demonstrated 3-4 fold higher transduction efficiency among human erythroid cells derived from transduced CD34+ cells in in vitro culture (34±0% vs 13±1%, p<0.01) and in xenografted mice (31±9% vs 7±5%, p<0.05). To evaluate transduction efficiency for long-term HSCs, we transduced rhesus CD34+ cells with our optimized reverse-oriented vector and our forward-oriented vector including GFP or YFP genes (instead of β-globin gene) in a competitive repopulation assay following 10 Gy total body irradiation. In two animals, GFP and YFP signals from both vectors were detected exclusively in red blood cells, documenting tissue specificity. Gene marking levels were 10-fold higher out to 4 years with the forward-oriented vector, compared to the reverse-oriented vector, and were comparable to standard GFP or YFP-marking vectors in 2 other animals. We then replaced the GFP gene with the β-globin gene containing intron 2 in the forward-oriented vector construct. To positively select intron-2-containing β-globin vectors, essential viral components (packaging signal, rev response element (RRE), or central polypurine tract (cPPT)) were deleted in the backbone of the forward-oriented vector, and the deleted viral components were inserted into intron 2 of the β-globin gene. We observed that half of the forward-oriented vectors lost intron 2 during vector preparation when no elements were included into intron 2. Insertion of the RRE resulted in positive selection of intron-2-containing β-globin vectors. We confirmed β-globin expression from the forward-oriented vector by hemoglobin A production in human erythroid cells derived from transduced peripheral blood mononuclear cells from SCD patients. Finally, human β-globin expression was detected in rhesus erythroid cells following transplantation of transduced CD34+ cells in 2 animals. In summary, we have developed a clinically relevant forward-oriented globin-expressing vector, which has 6 fold higher viral titers and 4-10 fold higher transduction efficiency for hematopoietic repopulating cells, as compared to the optimized reverse-oriented vector. RRE insertion allowed positive selection of intron-2-containing β-globin vectors, and human β-globin production was observed in transplanted rhesus macaques with the forward-oriented β-globin vector transduction. These findings bring us closer to a curative gene therapy for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

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