This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lotti, F. Articles by Ferrari, G. Search for Related Content PubMed PubMed Citation Articles by Lotti, F. Articles by Ferrari, G.

 Previous Article  |  Next Article 

Journal of Virology, April 2002, p. 3996-4007, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3996-4007.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Transcriptional Targeting of Lentiviral Vectors by Long Terminal Repeat Enhancer Replacement

Francesco Lotti,¹ Emilio Menguzzato,¹ Claudia Rossi,¹ Luigi Naldini,² Laurie Ailles,^2, Fulvio Mavilio,^3,4 and Giuliana Ferrari^1*

TIGET, Istituto Scientifico H. San Raffaele,¹ Genera S.p.A., 20132 Milan,⁴ Institute for Cancer Research and Treatment, University of Torino Medical School, 10060 Candiolo (Turin),² Department of Biomedical Sciences, University of Modena School of Medicine, 41100 Modena, Italy³

Received 9 August 2001/ Accepted 21 January 2002

Gene therapy of many genetic diseases requires permanent gene transfer into self-renewing stem cells and restriction of transgene expression to specific progenies. Human immunodeficiency virus (HIV)-derived lentiviral vectors are very effective in transducing rare, nondividing stem cell populations (e.g., hematopoietic stem cells) without altering their long-term repopulation and differentiation capacities. We developed a strategy for transcriptional targeting of lentiviral vectors based on replacing the viral long terminal repeat (LTR) enhancer with cell lineage-specific, genomic control elements. An upstream enhancer (HS2) of the erythroid-specific GATA-1 gene was used to replace most of the U3 region of the LTR, immediately upstream of the HIV type 1 (HIV-1) promoter. The modified LTR was used to drive the expression of a reporter gene (the green fluorescent protein [GFP] gene), while a second gene (a truncated form of the p75 nerve growth factor receptor [LNGFR]) was placed under the control of an internal constitutive promoter to monitor cell transduction, or to immunoselect transduced cells, independently from the expression of the targeted promoter. The transcriptionally targeted vectors were used to transduce cell lines, human CD34⁺ hematopoietic stem-progenitor cells, and murine bone marrow (BM)-repopulating stem cells. Gene expression was analyzed in the stem cell progeny in vitro and in vivo after xenotransplantation into nonobese diabetic-SCID mice or BM transplantation in coisogenic mice. The modified LTR directed high levels of transgene expression specifically in mature erythroblasts, in a TAT-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the modified LTR was higher, better restricted, and showed less position-effect variegation than that obtained by the same combination of enhancer-promoter elements placed in a conventional, internal position. Cloning of the woodchuck hepatitis virus posttranscriptional regulatory element at a defined position in the targeted vector allowed selective accumulation of the genomic transcripts with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of a spliced, major viral transcript to express a therapeutic gene and that of an internal, independently regulated promoter to express an additional gene for either cell marking or in vivo selection purposes.

---

* Corresponding author. Mailing address: TIGET-H. San Raffaele, Via Olgettina 58, 20132 Milan, Italy. Phone: 39-02-26434705. Fax: 39-02-26434668. E-mail: g.ferrari{at}hsr.it.

Present address: Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305.

---

Journal of Virology, April 2002, p. 3996-4007, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3996-4007.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Miccio, A., Cesari, R., Lotti, F., Rossi, C., Sanvito, F., Ponzoni, M., Routledge, S. J. E., Chow, C.-M., Antoniou, M. N., Ferrari, G. (2008). In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of {beta}-thalassemia. Proc. Natl. Acad. Sci. USA 105: 10547-10552 [Abstract] [Full Text]  
• Bonanomi, D., Menegon, A., Miccio, A., Ferrari, G., Corradi, A., Kao, H.-T., Benfenati, F., Valtorta, F. (2005). Phosphorylation of Synapsin I by cAMP-Dependent Protein Kinase Controls Synaptic Vesicle Dynamics in Developing Neurons. J. Neurosci. 25: 7299-7308 [Abstract] [Full Text]  
• Lucke, S., Grunwald, T., Uberla, K. (2005). Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors. J. Virol. 79: 9359-9362 [Abstract] [Full Text]  
• Davis, B. M., Humeau, L., Slepushkin, V., Binder, G., Korshalla, L., Ni, Y., Ogunjimi, E. O., Chang, L.-F., Lu, X., Dropulic, B. (2004). ABC transporter inhibitors that are substrates enhance lentiviral vector transduction into primitive hematopoietic progenitor cells. Blood 104: 364-373 [Abstract] [Full Text]  
• Testa, A., Lotti, F., Cairns, L., Grande, A., Ottolenghi, S., Ferrari, G., Ronchi, A. (2004). Deletion of a Negatively Acting Sequence in a Chimeric GATA-1 Enhancer-Long Terminal Repeat Greatly Increases Retrovirally Mediated Erythroid Expression. J. Biol. Chem. 279: 10523-10531 [Abstract] [Full Text]