Reduced Genotoxic Risk Using Foamy Viral Vectors for the Treatment of Canine Leukocyte Adhesion Deficiency

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3744-3744
Author(s):  
Thomas R. Bauer ◽  
Mehreen Hai ◽  
Rima L. Adler ◽  
James M. Allen ◽  
Laura M. Tuschong ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Leukocyte Adhesion ◽  
Severe Combined Immunodeficiency ◽  
Insertion Site ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Insertion Sites ◽  
Immunodeficiency Disease

Abstract Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.

scholarly journals Gene Therapies for Primary Immune Deficiencies

2021 ◽  
Vol 12 ◽  
Author(s):  
Lisa A. Kohn ◽  
Donald B. Kohn
Keyword(s):  
Gene Therapy ◽  
Leukocyte Adhesion ◽  
Severe Combined Immunodeficiency ◽  
Retroviral Vectors ◽  
Long Terminal Repeats ◽  
Hematopoietic Stem ◽  
Gene Therapies ◽  
Immune Deficiencies ◽  
Clinically Significant ◽  
Better Than

Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990–2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription (“SIN” vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs.

scholarly journals Preclinical Evaluation for the Gene Therapy of Patients with Leukocyte Adhesion Deficiency Type I

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5798-5798
Author(s):  
Elena Almarza ◽  
Cristina Mesa-Núñez ◽  
Carlos Damián ◽  
María Fernández-García ◽  
Begoña Díez-Cabezas ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Research Funding ◽  
Leukocyte Adhesion ◽  
Type I ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Deficiency Type

Abstract Leukocyte Adhesion Deficiency Type I (LAD-I) is a severe primary immunodeficiency characterized by recurrent and life-threatening bacterial infections. It is caused by mutations in the ITGB2 gene, encoding the integrin β2 common subunit (CD18). These mutations lead to defective or absent expression of β2 integrins on leukocyte surfaces, rendering leukocytes unable to extravasate to infection sites. Severe LAD-I is characterized by less than 2% of normal CD18 neutrophil expression and is fatal during the initial 2 years of life in 60-75% of patients in the absence of allogeneic hematopoietic transplant. As it is the case with other monogenic immunodeficiencies, LAD-I is a disorder that could be corrected by ex vivo gene therapy. To this aim we have developed a lentiviral vector (LV) that has recently obtained the Orphan Drug designation (EU/3/16/1753 and DRU-2016-5430). In this LV the expression of hCD18 is driven by a chimeric promoter with a higher activity in myeloid cells. Comprehensive safety and efficacy preclinical LV-mediated gene therapy studies have been conducted in LAD-I mouse models harboring either hypomorphic or knock-out mutations in the ITGB2 gene. Our studies demonstrate stable engraftment of gene corrected LAD-I mouse hematopoietic stem cells in LAD-I recipients and indicate a phenotypic correction of peripheral blood neutrophils. A complete preclinical safety evaluation of the vector was also carried out demonstrating the absence of hematotoxic and genotoxic effects in treated animals. Further studies have been conducted with GMP-produced LVs in human CD34+ cells aimed at optimization of cell transduction. The use of transduction enhancers (TEs) significantly improved the efficacy of genetic correction of human CD34+ cells transduced with LVs at low MOIs. Additionally, transplants into immunodeficient mice showed no changes in the repopulating ability of CD34+ cells when these cells were transduced in the presence of TEs. As occurred in in vitro cultures, significant increases in the transduction of repopulating cells were also associated with the use of TEs, indicating that optimized TE combinations will enable practical and cost-effective transduction of human HSCs in gene therapy protocols. Taken together, these results demonstrate the efficacy and safety of a gene therapy approach directed towards the therapy of LAD-I patients. Disclosures Almarza: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Bueren:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Safety Of a Gamma Globin Expressing Lentivirus Vector In a Non-Human Primate Model For Gene Therapy Of Sickle Cell Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2896-2896
Author(s):  
Hans-Peter Kiem ◽  
Paritha Arumugam ◽  
Burtner Christopher ◽  
Jennifer E Adair ◽  
Brian C Beard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Viral Vectors ◽  
Fetal Hemoglobin ◽  
Late Time ◽  
Hematopoietic Stem ◽  
Time Points ◽  
Insertion Sites ◽  
Time Point

Abstract Strategies for human gene therapy trials targeting hematopoietic stem cells (HSCs) are complicated by studies in murine models due to differences in stem cell behavior, short life-span and limited HSCs that could be transduced and transplanted when studying safety of viral vectors. Recent reports on adverse genotoxic events with integrating viral vectors in clinical trials utilizing autologous gene corrected HSCs underscores the need for safer gene transfer vectors. Non-human primates are relevant models due to similarities in the behavior of hematopoietic stem/progenitor cells, global gene expression profile, ability to assess long-term engraftment of transduced cells and safety of gene-modified HSCs, and thus could relatively accurately predict risk of vector genotoxicity. As a preclinical step towards globin gene therapy for hemoglobinopathies, we used pigtailed macaque HSC transplantation (HSCT) model to ascertain long-term safety and stable transgene expression from sGbG, a lentiviral vector (LV) encoding human γ-globin coding sequences from a β-globin promoter and locus control region (LCR). We observed upregulation of endogenous macaque fetal hemoglobin post-HSCT, which decreased to minimal levels by two years post-HSCT, a well-documented phenomenon following HSCT in humans. However, fetal hemoglobin (HbF) (comprised of macaque α and human γ-globin) expression remained steady at 12-15% even after 700 days post-HSCT. At 2.5 years post-HSCT, the HbF expression in a macaque transplanted with HSCs gene-modified with sGbG was stable in the range of 13% vs. 0.1% for control macaque; the average vector copy ranged between 0.13 and 0.28 with stable gene marking during the analysis period. In order to evaluate the LV integration site clonal population in sGbG transduced macaque repopulating cells, modified genome sequencing PCR was performed on genomic DNA from white blood cells and PCR products were sequenced. The junction sequences were mapped to the rhesus macaque genome assembly. A total of 177 unique vector insertions were retrieved at 6 months post-HSCT (early) and 102 vector insertions at 2.5 years (late) post-HSCT respectively. The relative distribution of vector insertions into chromosomes revealed a slight over-representation into Chromosome 16, both at early and late time points. Analysis of distribution of LV integrations of with respect to transcription start sites (TSS) revealed no insertions within the 2.5kb region of TSS. The frequency of insertions was concentrated near the 10-50kb window of TSS both upstream (18.6%) and downstream (15.6%) respectively. Interestingly, among the retrieved insertion sites, only 10% (17 insertions) were common at both time points, while 90% of insertions were unique at each time point, suggesting clonal fluctuations, with multiple HSC clones contributing to hematopoiesis at an early time point, and unique, HSC clones emerged at a later time point. Comparison of the top ten most frequently detected insertion sites at both time points revealed one insertion at Chromosome 16 mapping to an intron of KIAA0195 (an uncharacterized protein expressed ubiquitously), retrieved at both time points contributed to 3.27% and 9.23% of gene modified cells at early and late time points, respectively. No insertions were near MDS/EVI1, PRDM16 or HMGA2 loci. Other oncogenes and cancer associated genes were in the vicinity of some integrants; however, there was no significant clustering of insertions in gene regions. To assess the effect of insertions on flanking gene expression or putative cancer associated genes, we performed mRNAseq on whole blood RNA from sGbG macaque and two control macaques. A comparative analysis of transcript levels of >30,000 genes revealed no difference in global gene expression profile, gene insertions and genes within 300kb region of the LV insertion sites. Importantly, transcript levels of the most abundant clone observed (KIAA0195, Chr16: 70791901) and flanking genes, the tRNA splicing endonuclease subunit SEN54 and CASK interacting protein 2 differed from two control macaques analyzed by <2 fold. In summary, long-term follow-up data from a macaque that received cells gene-modified with a human γ-globin LV reveal polyclonal reconstitution of transduced cells, HSC clonal fluctuation, and a normal transcriptional profile, suggesting low risk of genotoxicity from this vector. Arumugam P, Burtner C: Equal Contribution 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.

scholarly journals Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3313-3320 ◽  
Author(s):  
Thomas R. Bauer ◽  
Mehreen Hai ◽  
Laura M. Tuschong ◽  
Tanya H. Burkholder ◽  
Yu-chen Gu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Leukocyte Adhesion ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Conditioning Regimens ◽  
Cell Gene ◽  
Stem Cell Gene

AbstractCanine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent, life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens—200 cGy total body irradiation (TBI) or 10 mg/kg busulfan—with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs, therapeutic levels of CD18+ leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment, and immunosuppression was not required for efficacy. The percentage of CD18+ leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification–mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific, large animal model using 2 clinically applicable conditioning regimens, and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD.

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