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Titolo:
DELAYED TARGETING OF CYTOKINE-NONRESPONSIVE HUMAN BONE-MARROW CD34(-MEDIATED GENE-TRANSFER ENHANCES TRANSDUCTION EFFICIENCY AND LONG-TERM EXPRESSION OF TRANSDUCED GENES() CELLS WITH RETROVIRUS)
Autore:
VEENA P; TRAYCOFF CM; WILLIAMS DA; MCMAHEL J; RICE S; CORNETTA K; SROUR EF;
Indirizzi:
INDIANA UNIV,SCH MED,DEPT MED,DIV HEMATOL ONCOL INDIANAPOLIS IN 46202 INDIANA UNIV,SCH MED,DEPT MED,DIV HEMATOL ONCOL INDIANAPOLIS IN 46202 INDIANA UNIV,SCH MED,DEPT MED,INDIANA ELKS CANC RES CTR INDIANAPOLIS IN 46202 INDIANA UNIV,SCH MED,HOWARD HUGHES MED INST,DEPT PEDIAT,HERMAN B WELLS CTR PEDIAT RES INDIANAPOLIS IN 46202 INDIANA UNIV,SCH MED,DEPT MICROBIOL & IMMUNOL INDIANAPOLIS IN 46202
Titolo Testata:
Blood
fascicolo: 10, volume: 91, anno: 1998,
pagine: 3693 - 3701
Fonte:
ISI
Lingua:
ENG
Soggetto:
HEMATOPOIETIC PROGENITOR CELLS; EX-VIVO EXPANSION; STEM-CELLS; PERIPHERAL-BLOOD; CORD-BLOOD; EXVIVO EXPANSION; THERAPY; CULTURE; TRANSPLANTATION; MARKING;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
49
Recensione:
Indirizzi per estratti:
Citazione:
P. Veena et al., "DELAYED TARGETING OF CYTOKINE-NONRESPONSIVE HUMAN BONE-MARROW CD34(-MEDIATED GENE-TRANSFER ENHANCES TRANSDUCTION EFFICIENCY AND LONG-TERM EXPRESSION OF TRANSDUCED GENES() CELLS WITH RETROVIRUS)", Blood, 91(10), 1998, pp. 3693-3701

Abstract

Primitive hematopoietic progenitor cells (HPCs) are potential targetsfor treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34(+) cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol using neo(R)-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34(-) cells. BM CD34(-) cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviralvector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and de CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR andd5 CNR cells and then infected separately with G1Na. Both sets of d5 and de CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in culturesinitiated with d5 and de CNR cells compared with respective CR fractions (P < .05), At week 2, transduction efficiency was comparable between d5 and de transduced OR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and de CNR fractions generated significantly higher numbers of neo(R) progenitor cells relative to the respective OR fractions (P < .05), while no difference in transduction efficiency between d5 and de CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transduced neo(R) gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the developmentof clinical gene therapy protocols. (C) 1998 by The American Society of Hematology.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 11/07/20 alle ore 21:06:04