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.

scholarly journals Correction of canine X-linked severe combined immunodeficiency by in vivo retroviral gene therapy

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3091-3097 ◽  
Author(s):  
Suk See Ting–De Ravin ◽  
Douglas R. Kennedy ◽  
Nora Naumann ◽  
Jeffrey S. Kennedy ◽  
Uimook Choi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Large Animal Model ◽  
Large Animal ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem

AbstractX-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor γ chain (γc). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term–surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.

scholarly journals Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 549
Author(s):  
Laura Garcia-Perez ◽  
Anita Ordas ◽  
Kirsten Canté-Barrett ◽  
Pauline Meij ◽  
Karin Pike-Overzet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Severe Combined Immunodeficiency ◽  
Good Manufacturing Practice ◽  
Proof Of Concept ◽  
Preclinical Development ◽  
Hematopoietic Stem ◽  
Immune Deficiencies ◽  
Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

scholarly journals Intratracheal aerosolization of viral vectors to newborn pig airways

BioTechniques ◽  
2020 ◽  
Vol 68 (5) ◽  
pp. 235-239
Author(s):  
Ashley L Cooney ◽  
Patrick L Sinn
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Endotracheal Tube ◽  
Viral Vectors ◽  
Small Animal ◽  
Large Animal ◽  
Large Animal Models ◽  
Airway Diseases ◽  
Efficient Delivery ◽  
Large Animals

Gene therapy for airway diseases requires efficient delivery of nucleic acids to the airways. In small animal models, gene delivery reagents are commonly delivered as a bolus dose. However, large animal models are often more relevant for the transition from preclinical studies to human trials. Aerosolizing viral vectors to the lungs of large animals can maximize anatomical distribution. Here, we describe a technique for aerosolization of viral vectors to the airways of newborn pigs. Briefly, a pig is anesthetized and intubated with an endotracheal tube, and a microsprayer is passed through the endotracheal tube. A fine mist is then sprayed into the distal trachea. Widespread and uniform distribution of transgene expression is critical for developing successful lung gene therapy treatments.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3216-3226 ◽  
Author(s):  
Aisha V. Sauer ◽  
Emanuela Mrak ◽  
Raisa Jofra Hernandez ◽  
Elena Zacchi ◽  
Francesco Cavani ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Severe Combined Immunodeficiency ◽  
Intrinsic Defect ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Gene therapy comes of age

Science ◽  
2018 ◽  
Vol 359 (6372) ◽  
pp. eaan4672 ◽  
Author(s):  
Cynthia E. Dunbar ◽  
Katherine A. High ◽  
J. Keith Joung ◽  
Donald B. Kohn ◽  
Keiya Ozawa ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
The United States ◽  
Genetically Engineered ◽  
Drug Status ◽  
Hematopoietic Stem ◽  
Gene Therapies ◽  
Engineered T Cells ◽  
Near Future

After almost 30 years of promise tempered by setbacks, gene therapies are rapidly becoming a critical component of the therapeutic armamentarium for a variety of inherited and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, eye and neurodegenerative disorders, and lymphoid cancers recently progressed to approved drug status in the United States and Europe, or are anticipated to receive approval in the near future. In this Review, we discuss milestones in the development of gene therapies, focusing on direct in vivo administration of viral vectors and adoptive transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss emerging genome editing technologies that should further advance the scope and efficacy of gene therapy approaches.

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.

Foamy Viral Vectors Efficiently Transduce Quiescent Hematopoietic Stem/Progenitor Cells (HSC) and Restore the Long Term Repopulating Activity of Fancc −/− Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 182-182 ◽  
Author(s):  
D. Wade Clapp
Keyword(s):  
Stem Cells ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Foamy Virus ◽  
Reporter Construct ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Increased Risk ◽  
Ex Vivo Culture

Abstract Fanconi anemia (FA) is characterized by bone marrow aplasia and myeloid leukemia. The identification of FA genes raises the potential of using gene transfer technology to introduce cDNAs into autologous HSCs. Current strategies using Moloney retroviruses require a 2–4 day ex vivo culture of HSC to facilitate stable integration of the transgene. However, ex vivo culture results in a time-dependent increase in apoptosis of Fancc−/− primitive HSC and mice reconstituted with the surviving cells have an increased risk of acquiring myeloid malignancies. Therefore we examined the potential of a recombinant foamy virus construct (MD9-FANCC-EGFP) to transduce murine Fancc −/− HSC in the absence of prestimulation. Forty-80% of progenitors that were in G0 – G1 at the time of transduction were transduced following a single 10–14 hr transduction. Aliquots of MD9-FANCC-EGFP transduced BM cells or cells encoding the EGFP transgene only were transplanted into irradiated recipient mice or recipients treated with IFN-g only. Four-six months following transplantation, recipient BM cells were isolated and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. Fancc−/− progenitors encoding recombinant FANCC were found to have a similar resistance to MMC as wildtype (WT) controls while Fancc−/− progenitors encoding the reporter construct only retained a high sensitivity to MMC. To assess the potential of MD9-FANCC-EGFP to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. The repopulating activity of MD9-FANCC-EGFP-transduced Fancc−/− stem cells was comparable to WT controls 18–24 months following transplantation in primary and secondary recipients. Additionally, while mice reconstituted with Fancc−/− cells transduced with the reporter construct had reduced repopulating ability as compared to the other groups, none of these recipients acquired myeloid malignancies. Collectively, these data provide in vivo evidence that an abbreviated transduction protocol utilizing a foamy-viral based vector allows efficient transduction of Fancc−/− HSC, and diminishes the selection pressure that occurs during ex vivo culture of Fancc−/− HSCs.

Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 997-1002 ◽  
Author(s):  
Tobias Neff ◽  
Brian C. Beard ◽  
Laura J. Peterson ◽  
Ponni Anandakumar ◽  
Jesse Thompson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Drug Resistance ◽  
Resistance Gene ◽  
Repeated Administration ◽  
Canine Model ◽  
Large Animal Model ◽  
Large Animal ◽  
Drug Resistance Gene ◽  
Methylating Agents

AbstractIncorporation of drug resistance genes into gene vectors has 2 important roles in stem cell gene therapy: increasing the proportion of gene-corrected cells in vivo (ie, in vivo selection) and marrow protection to permit higher or more tightly spaced doses of chemotherapy in the treatment of malignant diseases. We studied in a clinically relevant canine model of gene therapy the P140K mutant of the drug resistance gene methylguanine methyltransferase (MGMT), which encodes a DNA-repair enzyme that confers resistance to the combination of the MGMT inhibitor O6-benzylguanine (O6BG) and nitrosourea drugs such as carmustine and methylating agents such as temozolomide. Two dogs received MGMT(P140K)–transduced autologous CD34+-selected cells. After stable engraftment, gene marking in granulocytes was between 3% and 16% in the 2 animals, respectively. Repeated administration of O6BG and temozolomide resulted in a multilineage increase in gene-modified repopulating cells with marking levels of greater than 98% in granulocytes. MGMT(P140K) overexpression prevented the substantial myelosuppression normally associated with this drug combination. Importantly, hematopoiesis remained polyclonal throughout the course of the study. Extrahematopoietic toxicity was minimal, and no signs of myelodysplasia or leukemia were detected. These large-animal data support the evaluation of MGMT(P140K) in conjunction with O6BG and temozolomide in clinical trials.

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.

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