A Non-Human Primate Model To Study Anti-HIV Gene Therapy Strategies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3046-3046
Author(s):  
Grant D. Trobridge ◽  
Karen Beagles ◽  
Brian Beard ◽  
Ming-Jie Li ◽  
Jiing-Kuan Yee ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Foamy Virus ◽  
Large Animal ◽  
Primate Model ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Therapy Strategies ◽  
Anti Hiv ◽  
Human Primate

Abstract AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We are developing methylguanine-DNA-methyltransferase (MGMT) selection strategies for HSCs in the primate model to allow for high level marking with vectors containing anti-SHIV/HIV transgenes. We have obtained marking levels over 90% in granulocytes and over 30% in lymphocytes. To determine the effectiveness of an anti tat/rev shRNA to inhibit SHIV in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV. Expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR and replication of SHIV was inhibited as evidenced by inhibition of p27 production. Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (~45%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (~55%). Thus M. nemestrina monkeys provide a powerful model to test lenti and foamy virus mediated anti-HIV gene therapy strategies.

A Non-Human Primate Model for Lentivirus-Mediated Anti-HIV RNAi Strategies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3103-3103
Author(s):  
Karen Beagles ◽  
Brian Beard ◽  
John Rossi ◽  
Jiing-Kuan Yee ◽  
Shiu-lok Hu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Cell Line ◽  
Lentiviral Vector ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Hiv Tat ◽  
Cd34 Cells ◽  
Anti Hiv ◽  
Human Primate

Abstract AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lentiviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV89.6 P is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV89.6P results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We present here data showing efficient transduction of M. nemestrina CD34+ cells with an HIV-based lentiviral vector and RNAi-mediated inhibition of SHIV89.6 P replication in a hybrid T/B lymphocyte cell line (CEMx174). Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (» 50%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (» 60%). To determine effectiveness of anti tat/rev shRNA to inhibit SHIV89.6P in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene at MOIs of 1.3 and 3 respectively. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV 89.6P. One week post challenge, expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR. In summary, we have shown efficient HIV-based lentiviral transduction of M. nemestrina cells and efficient inhibition of SHIV infection by shRNA against HIV tat and rev thus providing a useful model to test lentiviral-mediated anti-HIV RNAi stem cell gene therapy in vivo.

A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 692-692
Author(s):  
Naoya Uchida ◽  
Phillip W Hargrove ◽  
Kareem Washington ◽  
Coen J. Lap ◽  
Matthew M. Hsieh ◽  
...  
Keyword(s):  
T Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Autologous Transplantation ◽  
Vector System ◽  
Transduction Efficiency ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 692 HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press). To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation. These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. Disclosures: No relevant conflicts of interest to declare.

Initial Results from the Northstar Study (HGB-204): A Phase 1/2 Study of Gene Therapy for β-Thalassemia Major Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral βΑ-T87Q -Globin Vector (LentiGlobin BB305 Drug Product)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 549-549 ◽  
Author(s):  
Alexis A. Thompson ◽  
John E Rasko ◽  
Suradej Hongeng ◽  
Janet L. Kwiatkowski ◽  
Gary Schiller ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Drug Product ◽  
Thalassemia Major ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Background: Hematopoietic stem cell (HSC) gene therapy has the potential to induce globin production and mitigate the need for blood transfusions in β-thalassemia major. Promising early results for 2 subjects with β0/βE -thalassemia major in the ongoing HGB-205 study suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered β-globin gene (βA-T87Q) can be safe and yield robust production of βA-T87Qglobin resulting in rapid transfusion independence. The Northstar study (HGB-204), which uses the same lentivirus vector and analogous study design as study HGB-205, is multi-center and multi-national, and centralizes drug product manufacturing. Herein, we provide the initial data on subjects enrolled and treated in this study. Subjects and Methods: Transfusion-dependent subjects with β-thalassemia major undergo HSC collection via mobilized peripheral blood apheresis and CD34+ cells are selected. Estimation of the mean ex-vivo vector copy number (VCN) is obtained by quantitative PCR performed on pooled colony-forming progenitors. Subjects undergo myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects are monitored for hematologic engraftment, βA-T87Q -globin expression (by high performance liquid chromatography) and transfusion requirements. Integration site analysis (ISA, by linear amplification-mediated PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus (RCL) assays are performed for safety monitoring. Results: As of 31 July 2014, 3 subjects have undergone HSC collection and ex-vivo LentiGlobin BB305 gene transfer. One subject (Subject 1102) has undergone myeloablation and drug product infusion. Outcomes data are shown in Table 1. The initial safety profile is consistent with myeloablation, without serious adverse events or gene therapy-related adverse events. This subject has increasing production of βA-T87Q-globin: the proportion of βA-T87Qglobin was 1.5%, 10.9% and 19.5% of total Hb at 1, 2 and 3 months post-infusion, respectively. This subject received pRBCs on Day +14 following drug product infusion and required no further transfusions until a single unit of pRBC was transfused on Day +96 for a Hb of 8.6 g/dL and fatigue. Two additional subjects have undergone drug product manufacture and are awaiting transplantation. Safety data related to ISA and RCL assays are pending. Abstract 549. Table 1 Preliminary results of dosing parameters and transplantation outcomes Subject Age (years) and Gender Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product- related Adverse Events βA-T87Q-Hb at last follow-up visit /Total Hb (g/dL) VCN CD34+ cell dose (x106 per kg) 1102 18 F β0/βE 1.0/1.1a 6.5 Day +17 None 1.77/8.6 1104 21 F β0/βE 0.7/0.7a 5.4 P P P 1106 20 F β0/β0 1.5 12.3 P P P As of 31 July 2014; P, pending a If more than one drug product were manufactured, the VCN of each drug product lot is presented. Conclusion: The first subject treated on the Northstar study has safely undergone drug product infusion with autologous HSCs transduced with LentiGlobin BB305 lentiviral vector and is producing steadily increasing amounts of βA-T87Q-globin. Additional follow-up of this subject plus data on additional subjects who undergo drug product infusion will be presented at the meeting. Ex-vivo gene transfer of βA-T87Q-globin to autologous HSCs is a promising approach for the treatment of patients with β-thalassemia major. Disclosures Thompson: ApoPharma: Consultancy; Novartis: Consultancy, Research Funding; Amgen: Research Funding; Glaxo Smith Kline: Research Funding; Mast: Research Funding; Eli Lilly: Research Funding. Kwiatkowski:Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy. Schiller:Sunesis, Amgen, Pfizer, Bristol Myers Squibb: Research Funding. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Petrusich:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment. Walters:Via Cord and AllCells, Inc.: Medical Director Other.

scholarly journals Foamy virus–mediated gene transfer to canine repopulating cells

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Hans-Peter Kiem ◽  
James Allen ◽  
Grant Trobridge ◽  
Erik Olson ◽  
Kirsten Keyser ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Blood Cells ◽  
Foamy Virus ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Cell Gene ◽  
Stem Cell Gene ◽  
Stem Cell Gene Therapy

AbstractFoamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)–expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/μL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP+, and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical.

Transduction of Macaque Hematopoietic Repopulating Cells with Lenti and Foamy Retroviral Vectors with MGMT Selection Cassettes To Evaluate AIDS Gene Therapy Strategies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3273-3273
Author(s):  
Grant D. Trobridge ◽  
Brian C. Beard ◽  
David Dickerson ◽  
Christina Gooch ◽  
Philip Olsen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Peripheral Blood ◽  
Retroviral Vectors ◽  
Hiv Replication ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Therapy Strategies ◽  
Anti Hiv

Abstract AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNAi in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. However inefficient gene delivery to hematopoietic stem cells has limited progress for AIDS gene therapy. We have developed both lenti and foamy retroviral vectors that contain methylguanine-DNA-methyltransferase (MGMT) expression cassettes to allow for in vivo selection, and have transduced macaque (M. nemestrina) long term repopulating cells with both vector systems. Following transplantation we observed rapid engraftment and levels of gene marking in the peripheral blood that should allow us to in vivo select both lenti and foamy-marked hematopoietic repopulating cells. In one animal transplanted with a lentiviral vector we obtained marking at 265 days post-transplant of over 30% in peripheral blood granulocytes and 20% in peripheral blood lymphocytes prior to in vivo selection. Anti-SHIV/HIV transgene cassettes targeting tat and rev that allow for potent inhibition of SHIV and HIV replication in vitro have been incorporated into both lenti and foamy vectors and we have transduced macaque long term repopulating cells with lenti vectors containing an anti-HIV cassette. We are currently developing protocols for efficient in vivo selection and future studies will investigate the ability of macaque hematopoietic repopulating cells transduced with lenti and foamy MGMT anti-HIV vectors to inhibit SHIV infection ex vivo and in vivo.

Generation Of FANCA-/- Human CD34+ Hematopoietic Stem Cells By shRNA Knockdown

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2903-2903
Author(s):  
Zejin Sun ◽  
Rikki Enzor ◽  
Paula Rio ◽  
D. Wade Clapp ◽  
Helmut Hanenberg
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vector ◽  
Human Fibroblasts ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Control Shrna ◽  
Cd34 Cells

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In-Vivo Gene Therapy with Foamy Virus Vectors

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1091 ◽  
Author(s):  
Yogendra Singh Rajawat ◽  
Olivier Humbert ◽  
Hans-Peter Kiem
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Foamy Virus ◽  
Severe Combined Immunodeficiency ◽  
Canine Model ◽  
Stable Gene ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Large Animal Models

Foamy viruses (FVs) are nonpathogenic retroviruses that infect various animals including bovines, felines, nonhuman primates (NHPs), and can be transmitted to humans through zoonotic infection. Due to their non-pathogenic nature, broad tissue tropism and relatively safe integration profile, FVs have been engineered as novel vectors (foamy virus vector, FVV) for stable gene transfer into different cells and tissues. FVVs have emerged as an alternative platform to contemporary viral vectors (e.g., adeno associated and lentiviral vectors) for experimental and therapeutic gene therapy of a variety of monogenetic diseases. Some of the important features of FVVs include the ability to efficiently transduce hematopoietic stem and progenitor cells (HSPCs) from humans, NHPs, canines and rodents. We have successfully used FVV for proof of concept studies to demonstrate safety and efficacy following in-vivo delivery in large animal models. In this review, we will comprehensively discuss FVV based in-vivo gene therapy approaches established in the X-linked severe combined immunodeficiency (SCID-X1) canine model.

Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector in X-Linked Adrenoleukodystrophy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 821-821 ◽  
Author(s):  
Marina Cavazzana-Calvo ◽  
Nathalie Cartier ◽  
Salima Hacein-Bey Abina ◽  
Gabor Veres ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that: ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term; no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed. (This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

Increasing Transduction Efficiency in Human Hematopoietic Stem Cells for Gene Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2052-2052
Author(s):  
Kismet M Baldwin ◽  
Fabrizia Urbinati ◽  
Zulema Romero-Garcia ◽  
Donald B. Kohn
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Clinical Trials ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Lentiviral Vector ◽  
Transduction Efficiency ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2052 Background: Sickle cell disease (SCD) is a multisystem disease, associated with severe episodes of acute illness and progressive organ damage. Currently, the only curative treatment is allogeneic hematopoietic stem cell transplant (HSCT); however, this is limited by availability of HLA compatible donors and by immunological complications of graft rejection or graft-versus-host disease. Autologous stem cell gene therapy for SCD has the potential to treat this illness without the immune suppression needed for current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levasseuer Blood 102:4312–9, 2003). Initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with the βAS3lentiviral (LV) vector for gene therapy of sickle cell disease has been demonstrated in our lab. However, this complex lentiviral vector is produced at a sub-optimal titer and large production batches would be needed to supply clinical trials. Hypothesis: Although, it has been proven that the βAS3 gene can be transduced into CD34+ hematopoietic stem/progenitor cells (HSPC), the transduction efficiency is still not optimal. The CD34+ cell population includes rare long-lived stem cells but also more abundant progenitors, which would be short-lived after transplant. We hypothesize that isolating the more primitive HSPC population (CD34+/CD38− cells approximately 1% of all CD34+ cells) and transducing them with the βAS3 lentiviral vector will increase transduction efficiency and greatly reduce vector needs. Methods: CD34+/CD38− cells were isolated from cord blood (CB) CD34+ cells obtained from healthy donors by fluorescence activated cell sorting (FACS) and transduced with the CCL.βAS3.FB LV vector. After 14 days in culture, vector copy number (VCN) was determined by qPCR. Isolation of a more primitive cell was confirmed via long term culture (LTC) assay for 90 days. At 2–3 weeks intervals, non-adherent cell number was obtained, VCN was analyzed and CFU assays were performed to assess their capability to fully maintain their hematopoietic potential after transduction. Results: CD34+/CD38− cells were effectively isolated using FACS (n=7; 6,329–33,742 cells; 34–99% theoretical yield). The isolated CD34+/CD38- cells were able to generate progeny over an extended period of LTC compared to the CD34+ cells whose cell expansion declined ∼60 days in culture. CFU assays demonstrated that βAS3 gene-modified CB CD34+/CD38- cells were fully capable of maintaining their hematopoietic potential. The isolated CD34+/CD38- cells required 3–40 fold less vector for transduction compared to an equivalent number of these cells contained within the larger, non-fractionated CD34+ preparations. Transduction of CD34+/CD38- cells measured at day 14, by qPCR, was improved relative to CD34+ cells, mean VCN 2.5, +/− SEM 0.33 (range 2–3.5) vs. VCN 1.3, +/− 0.40 (range 0.5–2), respectively (p=0.03). In LTC, VCN remained higher over time in the CD34+/CD38- cells compared to the CD34+ cells, mean VCN 2.0, +/− SEM 0.13 (range 1.6–2.7) vs. VCN 0.5, +/− 0.09 (range 0.2–0.9) respectively. In vivo studies are ongoing to investigate the transduction efficiency of stem/progenitor cells engrafting from CD34+ and CD34+/CD38- cells transplanted in the NSG mouse model. Immunomagnetic isolation of CD34+/CD38- cells using columns is underway in anticipation of potential use in future clinical trials. Further investigations into the mechanisms for increased transduction in the CD34+/CD38- cells are ongoing. Conclusions: This work provides initial evidence for the beneficial effects from isolating human CB CD34+/CD38− cells to improve transduction with the βAS3LV vector for gene therapy of sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

Study Hgb-205: Outcomes of Gene Therapy for Hemoglobinopathies Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral βΑ-T87Q-Globin Vector (LentiGlobin® BB305 Drug Product)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4797-4797 ◽  
Author(s):  
Marina Cavazzana ◽  
Jean-Antoine Ribeil ◽  
Emmanuel Payen ◽  
Felipe Suarez ◽  
Yves Beuzard ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Drug Product ◽  
Thalassemia Major ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Equity Ownership

Abstract Background: In patients with β-thalassemia major, hematopoietic stem cell (HSC) gene therapy has the potential to induce production of β-globin, γ-globin or modified β-globin in the red blood cell lineage and reduce or stop the need for blood transfusions. We have previously presented early results for 2 subjects with β0/βE -thalassemia major that suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered β-globin gene (βA-T87Q) resulted in near-normal levels of total hemoglobin (Hb) early after HSC infusion. Herein, we provide additional follow-up data on these two subjects. Subjects and Methods: After obtaining informed consent, subjects with β-thalassemia major underwent HSC collection via peripheral blood apheresis and CD34+ cells were selected. Estimation of the mean ex- vivo vector copy number (VCN) was obtained by quantitative PCR performed on pooled colony-forming progenitors. Subjects underwent myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects were monitored for hematological engraftment, βA-T87Q-globin expression (by high performance liquid chromatography) and transfusion requirements. Integration site analysis (ISA, by linear amplification-mediated PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus (RCL) assays were performed. Results: As of 31 July 2014, two subjects with β0/βE thalassemia major (Subjects 1201 and 1202) have undergone infusion with drug product. The outcome of these two subjects to date is shown in Table 1. The initial safety profile is consistent with myeloablation, without serious adverse events or drug product-related adverse events. Both subjects remain transfusion independent. ISA analyses in both the subjects at 3 months shows polyclonal reconstitution. An additional 2 subjects have been enrolled in this study but have not yet undergone drug product infusion. Conclusion: In the first two subjects, early transfusion independence was achieved and has been maintained as of 31 July 2014. Further follow up data on these two subjects and additional data on subjects who have undergone drug product infusion in this study will be presented. Gene therapy using autologous HSC transduced with LentiGlobin BB305 lentiviral vector is a promising approach for the treatment of patients with β-thalassemia major. Abstract 4797. Table 1. Preliminary Results of Dosing Parameters and Transplantation Outcomes Subject Age (years) and gender Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product-related Adverse Events Day of last pRBC transfusion Day of last follow up βA-T87Q-Hb at last follow-up visit /Total Hb (g/dL) VCNa CD34+ cell dose (x106 per kg) 1201 19 F β0/βE 1.5 8.9 Day +13 None Day +10 Day +180 7.2/10.2 1202 16 M β0/βE 2.1 13.6 Day +15 None Day +12 Day +90 6.8/11.0 As of 31 July 2014 a VCN, mean vector copy number Disclosures Payen: bluebird bio, Inc: Consultancy. Beuzard:bluebird bio, Inc: Consultancy, Equity Ownership. Sandler:bluebird bio, Inc: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership. De Montalembert:Novartis : Speakers Bureau. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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