826. Transduction of Human Hematopoietic Stem Cells by RD114-TR-Pseudotyped Lentiviral Vectors

HIV-1-derived lentiviral vectors are efficiently pseudotyped by a chimeric envelope (RD114-TR) encoding the extracellular and transmembrane domains of the FLV RD114 glycoprotein fused to cytoplasmic tail (TR) of the MLV 4070A amphotropic glycoprotein. RD114-TR pseudotyped vectors may be concentrated by centrifugation, are resistant to complement inactivation, and are of particular interest for both ex vivo and in vivo gene therapy applications. We carried out a comparative analysis of VSV-G and RD114-TR-pseudotyped lentiviral vectors in transducing human cord blood-derived CD34+ hematopoietic stem/progenitor cells. Transduction efficiency was comparatively analysed in CD34+ cells in liquid culture, in the progeny of CD34+ clonogenic progenitors in semi-solid culture, and in the progeny of CD34+ repopulating stem cells after xeno-transplantation in NOD-SCID mice. In all cases, RD114-TR-pseudotyped vectors transduced hematopoietic cells at lower m.o.i., resulting in lower toxicity and more efficient stable transduction at comparable vector copy number per genome. Potential changes in CD34+ cells transcription profile and phenotype upon transduction with RD114-TR or VSV-G-pseudotyped vectors was investigated by Affymetrix Gene Chips microarray analysis. We found no significant difference in gene expression patterns between mock-RD114-TR and VSV-G-transduced cells. Our study show that the biology of repopulating hematopoietic stem cells and their progeny is not affected by transduction with RD114-TR-pseudotyped lentiviral vectors

Stem cell plasticity: time for a reappraisal?

n recent years an increasing number of publications have claimed that adult mammalian stem cells (SC) may be capable of differentiating across tissue lineage boundaries and that this plasticity may represent a novel therapeutic strategy for tissue regeneration. However, after a first phase of excitement, the issue of somatic SC plasticity remains controversial and the therapeutic perspectives are still elusive. In this review, we examine the general mechanisms which govern the function of SC, the identification and functional characterization of adult SC of different tissues and their putative capacity to transdifferentiate into mature cells of different origin. The potential clinical applications of adult SC for regenerative medicine are also discussed in each chapter. The method employed for preparing this review was the informal consensus development. Members of the Working Group on SC met four times and discussed the single points, previously assigned by the Chairman (S.T.), in order to achieve an agreement on different opinions and approve the final manuscript. All the authors of the present review have been working in the field of SC and have contributed original papers to peer-reviewed journals. In addition to the authors' own work, the present review examines articles published in journals covered by the Science Citation Index and Medline

Preclinical corrective gene transfer in Xeroderma pigmentosum human skin stem cells

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (> 10(40) cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients.F.L. was supported in part by grants PI081054 from ISCIII and PBIO-0306-2006 from Comunidad de Madrid (CAM). M.D.R. was supported by grant SAF2010-16976 from MICINN. The authors declared no conflict of interest

Estimated Comparative Integration Hotspots Identify Different Behaviors of Retroviral Gene Transfer Vectors

Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas

Selective engraftment of genetically modified hematopoietic stem cells by a truncated erythropoietin receptor

Genetic modification of hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Transplantation of HSCs, however, requires toxic myeloablation regimens which render this approach questionable for non life-threatening disorders. A potential alternative is the use of transgenes allowing positive selection of HSCs in vivo. We used MLV-derived retroviral vectors and HIV-derived lentiviral vectors to express a truncated form of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. The tEpoR molecule carries a deletion of the 91 carboxy-terminal amino acids, which enhances its proliferative response due to the elimination of a negative regulatory domain. Murine HSCs expressing retrovirally-transferred tEpoR at different levels (1,500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into co-isogenic mouse recipients. Human cord blood-derived CD34+ stem/progenitor cells transduced with a lentiviral vector expressing tEpoR significantly increase their marrow repopulation capacity upon xenotransplantation into sub-lethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival and differentiation properties. Long-term analysis of serially transplanted mice showed that expression of tEpoR at physiological levels (i.e., comparable with, or slightly higher than, those of the wild-type EpoR in erythroblastic cells) has no effect on steady-state hematopoiesis, and induces no further expansion of transduced cells after the engraftment period. However, significant overexpression of tEpoR (>8-fold the physiological levels) causes mild anemia and erythrocyte morphological abnormalities. These data indicate that expression of tEpoR is a potential alternative for in vivo selection of murine and human repopulating HSCs

Competitive engraftment of hematopoietic stem cells genetically modified with a truncated erythropoietin receptor

Transplantation of genetically modified hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Engraftment of HSCs, however, requires toxic myeloablative treatments, which render this approach questionable for non-life-threatening disorders. A potential alternative is the use of transgenes, which allows positive selection of HSCs in vivo. We used retroviral vectors to express a truncated derivative of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. Murine HSCs expressing tEpoR at different levels (1500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into fully or partially myeloablated co-isogenic mouse recipients. Long-term analysis of transplanted mice showed that expression of tEpoR at paraphysiological levels (similar to 1500 receptors/cell) has no effect on steady-state hematopoiesis and induces no further expansion of transduced cells after the engraftment period. Human cord blood-derived CD34(+) stem/progenitor cells transduced with a lentiviral vector expressing tEpoR expand their clonogenic capacity in vitro, and significantly increase their marrow repopulation capacity upon xenotransplantation into sublethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival, and differentiation properties. These data indicate that expression of tEpoR is an effective strategy to promote selective engraftment of genetically modified HSCs upon transplantation in both myeloablative and nonmyeloablative conditions, without the use of toxic drugs for selection

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

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self-inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [18] enhancers) and WPRE expressed at levels higher than the HS2/beta -promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34+ cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The 18/HS-40-containing vector encoding a hybrid human beta/gamma -globin gene led to 43% to 113% human gamma -globin expression/copy of the mouse alpha -globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to Identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases

Long-term engraftment of single genetically modified epidermal stem cell-derived clones enables safety pre-assessment of human cutaneous gene therapy

Predicting the risks of permanent gene therapy approaches involving the use of integrative gene-targeting vectors has become a critical issue after the unfortunate episode of a clinical trial in children with X-linked severe combined immunodeficiency (X-SCID). Safety pre-assessment of single isolated gene-targeted stem cells or their derivative clones able to regenerate their tissue of origin would be a major asset in addressing untoward gene therapy effects in advance. Human epidermal stem cells, which have extensive proliferative potential in vitro, theoretically offer such a possibility as a method of assessment. By means of optimized organotypic culture and grafting methods, we demonstrate the long-term in vivo regenerative capacity of single gene-targeted human epidermal stem cell clones (holoclones). Both histopathological analysis of holoclone-derived grafts in immunodeficient mice and retroviral insertion site mapping performed in the holoclone in vitro and after grafting provide proof of the feasibility of pre-assessing genotoxicity risks in isolated stem cells before transplantation into patients. Our results provide an experimental basis for previously untested assumptions about the in vivo behavior of epidermal stem cells prospectively isolated in vitro and pave the way for a safer approach to cutaneous gene therapy

Absence of an intrathecal immune reaction to a helper-dependent adenoviral vector delivered into the cerebrospinal fluid of non-human primates.

Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration