Ex Vivo Gene Trasfer for Hemophilia A That Enhances Safety and Results in Sustained In Vivo Factor VIII Expression from Lentivirally-Engineered Endothelial Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3251-3251
Author(s):  
Hideto Matsui ◽  
Masaru Shibata ◽  
Brian Brown ◽  
Andrea Labelle ◽  
Carol Hegadorn ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cell ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Scid Mice ◽  
Anatomic Site ◽  
Cmv Promoter ◽  
Endothelial Progenitors ◽  
Therapy Strategy

Abstract Hemophilia is an excellent candidate disorder for the use of gene therapy as a treatment modality. However, significant obstacles have been encountered with systemic delivery of viral vectors that have prevented sustained expression of the therapeutic protein. Investigation of alternative gene therapy strategies for hemophilia that enhance safety and facilitate long-term, therapeutic levels of the transgene product is imperative. In this study, we evaluated an ex vivo gene therapy strategy for hemophilia A. Circulating endothelial cell progenitors (blood outgrowth endothelial cells - BOECs) were isolated from canine and mouse blood and transduced with a third generation self-inactivating lentiviral vector encoding the canine FVIII transgene under the transcriptional control of either the CMV promoter or an endothelial cell-specific regulatory element. Transduced BOECs were injected either intravenously (IV) or subcutaneously mixed with Matrigel (SC+Matrigel) into NOD/SCID mice. Canine FVIII antigen levels were assayed at weekly intervals using an Asserachrom VIII:Ag ELISA that detects canine FVIII against a background of normal murine FVIII levels in the NOD/SCID mice. The mean FVIII antigen levels in mice injected with BOECs at 3 weeks following treatment were 37.5 mU/mL and 105.8mU/mL, for IV and SC+Matrigel administration, respectively. These FVIII antigen levels were sustained up to 12 weeks at therapeutic levels (21.3mU/mL and 21.7mU/mL, for IV and SC+Matrigel administration respectively). To evaluate if the observed loss of FVIII expression by 12 weeks post-treatment resulted from transcriptional silencing of the viral promoter, the CMV promoter was replaced with the endothelial cell-specific thrombomodulin (TM) promoter and transduced BOECs were implanted SC with Matrigel. In contrast to results from the CMV-regulated transgene, sustained therapeutic levels of FVIII have been documented for the duration of the study with the TM-regulated construct (34.3 mU/mL at 3 weeks and 22.5 mU/mL at 20 weeks) Immunostaining at 18 weeks after SC implantation of the transduced BOECs, shows that these cells still express FVIII and von Willebrand Factor. Biodistribution analysis by flow cytometry and quantitative PCR demonstrated that SC-implanted BOECs were retained inside the scaffold and were not detected at any other anatomic site. These results indicate that genetically-modified endothelial progenitors implanted in a SC scaffold can provide sustained therapeutic levels of FVIII and are a promising safe delivery vehicle for gene therapy of hemophilia. Currently, these engineered cells have been implanted into immunocompetant mice and FVIII levels are being assessed.

Genetically-Engineered Endothelial Cells Implanted Into the Omentum of Hemophilia A Dogs Provides Long-Term Circulating FVIII Resulting From Sustained FVIII Expression and Persistent Cell Viability.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3578-3578 ◽  
Author(s):  
Margareth C Ozelo ◽  
Hideto Matsui ◽  
Carol A. Hegadorn ◽  
Lori Harpell ◽  
Sandra Powell ◽  
...  
Keyword(s):  
Immune Response ◽  
Endothelial Cells ◽  
Growth Factors ◽  
Cell Viability ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Autologous Blood ◽  
Cell Transplant ◽  
Therapy Strategy

Abstract Abstract 3578 Poster Board III-515 Ex vivo delivery of therapeutic transgenes provides an additional level of safety as it avoids systemic administration of viral vectors. Our group has shown that autologous blood outgrowth endothelial cells (BOECs) transduced with a lentiviral vector delivery system containing the FVIII transgene is a promising gene therapy strategy for hemophilia A. We have shown that subcutaneous implantation of factor (F) VIII-expressing BOECs in a murine model of hemophilia A can produce therapeutic levels of FVIII that are sustained for more than 6 months. However, to improve the levels of FVIII expression and cell viability we wanted to evaluate the omentum as an alternative site for BOEC implantation. Initially this strategy was evaluated in two normal dogs. One and three months after delivery of the cells, immunostaining of biopsies from the injection sites showed the presence of viable cells expressing FVIII and evidence of neovascularization. To evaluate the efficacy of this strategy, a hemophilia A dog received 5×108 transduced autologous BOECs that expressed high levels of FVIII in vitro (1.5 IU/106 cells/24hrs). We used autologous fibrinogen as a vehicle for the cells along with canine endothelial growth factors (VEGF and bFGF). For the implantation procedure the dog received prophylaxis with canine cryoprecipitate transfusions. FVIII antigen levels (FVIII:Ag) of between 20 and 50 ng/mL continue to be detected in the plasma 8 months post-implantation, indicating that these cells remain viable and express persistent high levels of FVIII over an extended period of time. However, two weeks after the procedure, the dog developed an anti-FVIII immune response comprising both inhibitory and non-inhibitory antibodies, and therefore no FVIII coagulant activity (FVIII:C) was detected. With a view to preventing the development of the anti-FVIII immune response, we used immunosuppression with cyclophosphamide in two additional hemophilia A dogs. Each of these dogs received 5 × 108 transduced autologous BOECs. In place of fibrinogen, these cells were delivered in a gel comprised of synthetic, heparin-binding peptide-amphiphiles (HBPA) and heparan sulfate, along with canine VEGF and bFGF. The peptide gel prolongs the activity of these growth factors and protects them from proteolysis, enhancing their angiogenic activity. HBPA gel has been shown to increase vascularization of cell transplant sites, which should improve BOEC survival in the omentum. The procedure of implanting the genetically modified BOECs was completed without complications in all hemophilia A dogs and we are continuing to evaluate the efficacy of this strategy. Disclosures: Hulvat: Nanotope Inc.: Employment.

scholarly journals Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo Factor VIII Expression from Lentivirally Engineered Endothelial Progenitors

Stem Cells ◽  
2007 ◽  
Vol 25 (10) ◽  
pp. 2660-2669 ◽  
Author(s):  
Hideto Matsui ◽  
Masaru Shibata ◽  
Brian Brown ◽  
Andrea Labelle ◽  
Carol Hegadorn ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Ex Vivo ◽  
Safe Delivery ◽  
Endothelial Progenitors ◽  
Ex Vivo Gene Therapy

scholarly journals GLT1 gene delivery based on bone marrow-derived cells ameliorates motor function and survival in a mouse model of ALS

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Natsuko Ohashi ◽  
Tomoya Terashima ◽  
Miwako Katagi ◽  
Yuki Nakae ◽  
Junko Okano ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Lentiviral Vector ◽  
White Blood Cells ◽  
Glutamate Toxicity ◽  
Pathological Lesion ◽  
Arginase 1 ◽  
Therapy Strategy

AbstractAmyotrophic lateral sclerosis (ALS) is an intractable neurodegenerative disease. CD68-positive bone marrow (BM)-derived cells (BMDCs) accumulate in the pathological lesion in the SOD1(G93A) ALS mouse model after BM transplantation (BMT). Therefore, we investigated whether BMDCs can be applied as gene carriers for cell-based gene therapy by employing the accumulation of BMDCs. In ALS mice, YFP reporter signals were observed in 12–14% of white blood cells (WBCs) and in the spinal cord via transplantation of BM after lentiviral vector (LV) infection. After confirmation of gene transduction by LV with the CD68 promoter in 4–7% of WBCs and in the spinal cord of ALS mice, BM cells were infected with LVs expressing glutamate transporter (GLT) 1 that protects neurons from glutamate toxicity, driven by the CD68 promoter, which were transplanted into ALS mice. The treated mice showed improvement of motor behaviors and prolonged survival. Additionally, interleukin (IL)-1β was significantly suppressed, and IL-4, arginase 1, and FIZZ were significantly increased in the mice. These results suggested that GLT1 expression by BMDCs improved the spinal cord environment. Therefore, our gene therapy strategy may be applied to treat neurodegenerative diseases such as ALS in which BMDCs accumulate in the pathological lesion by BMT.

scholarly journals Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Tugba Mehmetoglu-Gurbuz ◽  
Rose Yeh ◽  
Himanshu Garg ◽  
Anjali Joshi
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Suicide Gene ◽  
Vector System ◽  
Tat Protein ◽  
Ccr5 Gene ◽  
Hematopoietic Stem ◽  
Therapy Strategy ◽  
Hiv Tat ◽  
A Cell

Abstract Background Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. Methods A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. Results Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. Conclusions Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

scholarly journals Lentiviral Vector-Mediated Gene Transfer Restores CD18 Expression in Long-Term Repopulating Hematopoietic Stem and Progenitor Cells Isolated from a Patient with Leukocyte Adhesion Deficiency Type 1

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1859-1859
Author(s):  
Richard H. Smith ◽  
Daisuke Araki ◽  
Andre Larochelle
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Leukocyte Adhesion ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ex Vivo Gene Therapy ◽  
Deficiency Type

Abstract Leukocyte adhesion deficiency type 1 (LAD-1) is an inherited primary immunodeficiency caused by loss-of-function mutation within the ITGB2 gene, which encodes the beta2 integrin subunit CD18. Individuals with LAD-1 experience significant loss of neutrophil-mediated innate cellular immune function, resulting in delayed wound healing, severe periodontitis, and life-long bouts of bacterial infection. LAD-1 is a prime candidate for lentiviral vector-mediated genetic intervention as i) it is an intractable, potentially life-threatening disease with limited treatment options, ii) it is amenable to current ex vivo gene therapy procedures, and iii) partial phenotypic correction would present a high likelihood of significant clinical benefit. Allogeneic stem cell transplant can be curative, but suffers from matched donor availability and the potential for graft-versus-host disease. Autologous ex vivo gene therapy may provide a viable alternative to allogeneic transplant in LAD-1 patients. We have evaluated the ability of a CD18-expressing lentiviral vector (LV-hCD18) to mediate ex vivo transduction of LAD-1 patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and subsequent long-term LAD-1 HSPC engraftment in immunodeficient NOD-scid IL2Rg null (NSG) mice. An open reading frame encoding human CD18 was placed under the transcriptional control of the MND promoter (a modified retroviral promoter associated with high levels of stable transgene expression) and packaged in VSV-G-pseudotyped lentiviral particles. After 1 day of pre-stimulation, LAD-1 HSPCs were transduced with LV-hCD18 (MOI = 10) in the presence or absence of transduction-enhancing adjuvants, poloxamer 407 (P407) and prostaglandin E2 (PGE 2), for 24 hours. Sublethally irradiated NSG mice (7 mice/group) were transplanted with either mock-transduced LAD-1 HSPCs, LAD-1 HSPCs transduced in the absence of adjuvants, or LAD-1 HSPCs transduced in the presence of P407/PGE 2. Bone marrow was harvested at ~5.5 months post-transplant for flow cytometric analyses of engraftment efficiency, transgene marking, and human blood cell lineage reconstitution. Bone marrow from mice that received mock-transduced LAD-1 HSPCs showed an average total of 6.45 ± 2.54% (mean ± SEM) CD45+ human cells. Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 7.99 ± 1.82% CD45+ human cells, whereas mice transplanted with LAD-1 HSPCs transduced in the presence of adjuvants showed 7.33 ± 1.90% CD45+ cells. A Kruskal-Wallis statistical test indicated no significant difference in the level of human cell engraftment among the recipient groups (P=0.72). Consistent with the LAD-1 phenotype, human myeloid cells from mice that received mock-transduced LAD-1 HSPCs displayed only background levels of CD18 marking (0.13 ± 0.06% CD45+CD13+CD18+ cells). Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 ± 0.40% CD18+ human myeloid cells (range 2.19% to 5.50%), whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE 2 showed 9.56 ± 0.96% CD18+ human myeloid cells (range 4.63% to 13.10%), thus representing a >2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. Moreover, vector-mediated expression of CD18 rescued endogenous expression of a major CD18 heterodimerization partner in neutrophils, CD11b. In mock-transduced LAD-1 HSPC recipients, CD13+ human myeloid cells were devoid of cell surface CD11b expression (0.01 ± 0.01% CD45+CD13+CD11b+ cells). In contrast, CD13+ human myeloid cells in mice that received LAD-1 HSPCs transduced in the absence of adjuvant showed detectable levels of CD11b expression (2.62 ± 0.19% of CD18-expressing human myeloid cells), and CD11b levels were increased to 6.90 ± 0.98% in LAD-1 HSPCs transduced in the presence of P407/PGE 2. Multilineage engraftment, as evidenced by the presence of CD3+ T cells and CD20+ B cells, was noted within all groups; however, human myeloid cells represented the most prominent human blood cell compartment observed. Colony-forming-unit assays of transduced cells and non-transduced control cells pre-transplant showed similar clonogenic output and colony diversity. In sum, successful transduction, engraftment, transgene marking, CD11b rescue, and multilineage reconstitution supports further development of lentiviral vector-mediated gene therapy for LAD-1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

High-Expression Porcine FVIII Driven by Erythroid-Specific Promoters for Lentiviral Vector-Mediated Gene Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3544-3544
Author(s):  
Nadia Sutherland ◽  
Kerry L Dooriss ◽  
David A McCarty ◽  
Christopher B Doering ◽  
H. Trent Spencer
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Blood Cells ◽  
Copy Number ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
K562 Cells ◽  
Dna Copy Number ◽  
Normal Human ◽  
Globin Promoter

Abstract Hemophilia A is an X-linked gene disorder that results in a deficiency of circulating coagulation factor VIII (fVIII) and may be ameliorated by only modest amounts of circulating protein, which makes it a logical candidate for gene therapy. Due to the potential risk of insertional mutagenesis from oncoretroviral-mediated gene therapy, cell-specific expression of transgenes using self-inactivating viral vectors may provide a safer gene therapy approach for use in humans. Therefore, we constructed simian immunodeficiency virus (SIV)-based lentiviral vectors containing a 5′ long-terminal repeat (LTR) and 3′ LTR with self-inactivating U3 deletion, the bovine growth hormone polyA signal, a packaging signal (ψ), and a single internal ankyrin-1 or β-globin promoter, designated SIV-Ank and SIV-Bg, respectively. The minimal 314-bp ankyrin-1 promoter and 180-bp β-globin promoter flanked upstream by enhancing sequences, HS2, HS3, and HS4 (Hanawa et al., Hum Gene Ther, 2002) from the locus control region were cloned into the SIV vector backbone upstream from either enhanced green fluorescent protein (eGFP) or B-domain deleted porcine factor VIII (BDDpfVIII). The erythroid-specificity of each promoter was evaluated in vitro by measurement of either eGFP or fVIII expression following transduction of SIV-Ank and SIV-Bg constructs into both K562 myelogenous leukemic cells and 293T human embryonic kidney cells. GFP expression, as measured by flow cytometry, in transduced cells revealed that the ankyrin-1 and β-globin promoters are more active in K562 cells as compared to 293T cells. The β-globin promoter yielded higher mean fluorescent intensity values for GFP compared to the ankyrin-1 promoter at similar MOIs in K562 cells, suggesting stronger β-globin promoter activity in these cells. Transduction of cells with the SIV vector encoding BDDpfVIII driven by the β-globin promoter resulted in a 14-fold higher number of transcripts per DNA copy number in K562 cells compared to 293T cells, while cells transduced with the ankyrin-l promoter had only a 1.4-fold greater number of transcripts per DNA copy number. In addition, SIV-Bg-fVIII-modified K562 cells produced a 5.2-fold greater number of transcripts per DNA copy number than SIV-Ank- fVIII-modified cells. To evaluate the usefulness of these vectors for in vivo expression of BDDpfVIII, hemophilia A mice (exon 16 knockout) were conditioned with 11 Gy total body irradiation and transplanted with gene-modified Sca-1+ cells transduced with either SIV-Ank-fVIII, SIV-Ank-eGFP, SIV-Bg-fVIII, or SIV-Bg-eGFP. The expression of eGFP from donor red blood cells in recipient mice was approximately 8–12% using both the ankyrin-1 and β-globin promoter constructs. Mice that received cells transduced with SIVAnk- fVIII demonstrated therapeutic levels of plasma fVIII up to 0.5 units/mL (i.e. 50% normal human levels). However, fVIII expression decreased over time and real-time PCR analysis of peripheral blood cells confirmed the loss of detectable fVIII transgene by 6 weeks after transplantation, suggesting there was predominantly gene transfer into short-term repopulating hematopoietic cells. Mice transplanted with SIV-Bg-fVIII-modified hematopoietic stem cells demonstrated a similar rise and fall of fVIII expression within the first 4 weeks after transplantation, and showed an increase in fVIII expression by 6 weeks. At 8 weeks post transplantation, fVIII levels greater than 300% normal human levels were observed. Red blood cell count, hemoglobin, and red blood cell morphology were normal despite the high level of expression of fVIII. Overall these data demonstrate the potential for therapeutic expression of factor VIII using a self-inactivating lentiviral vector containing an erythroid-specific internal promoter.

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