Sustained Long Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Third-Generation Lentiviral Vectors

doi:10.1634/stemcells.2008-0161

TISSUE-SPECIFIC STEM CELLS

Sustained Long Term Engraftment and Transgene Expression of Peripheral Blood CD34⁺ Cells Transduced with Third-Generation Lentiviral Vectors

Melania Tesio ¹, Loretta Gammaitoni ¹, Monica Gunetti ², Valeria Leuci ¹, Ymera Pignochino ¹, Noela Jordaney ¹, Sonia Capellero ¹, Cristina Cammarata ¹, Luisa Caione ¹, Giuseppe Migliaretti ³, Franca Fagioli ², Antonio Tabilio ⁴, Massimo Aglietta ¹, Wanda Piacibello ¹^*

¹ Laboratory of Clinical Oncology, Department of Oncological Sciences, University of Torino Medical School- IRCC, Institute for Cancer Research and Treatment, 10060 Candiolo-Torino Italy
² Pediatric Onco-Haematology Unit, Stem Cell Transplantation and Cellular Therapy Center, Regina Margherita Children's Hospital, Turin, Italy
³ Department of Public Health and Microbiology, University of Torino Medical School, Stem Cell Transplantation and Cellular Therapy Center, Regina Margherita Children's Hospital, Turin, Italy
⁴ Hematology and Clinical Immunology Section, and Internal Medicine and Oncological Science Section, University of Perugia, 06123 Perugia, Italy

^* To whom correspondence should be addressed. E-mail: wanda.piacibello{at}ircc.it.

Abstract

As mobilized peripheral blood (MPB) represents an attractivecell source for gene therapy, we investigated the ability ofthird-generation lentiviral vectors (LVs) to transfer the EnhancedGreen Fluorescent Protein (EGFP) gene into MPB CD34⁺ cells inculture conditions allowing expansion of transplantable humanhematopoietic stem cells (HSCs). To date, few studies have reportedtransduction of MPB cells with VSV-G pseudotyped LVs. The criticalissue remains whether primitive, hematopoietic repopulatingcells have, indeed, been transduced.

In vitro (5 weeks' culturein FL+TPO+SCF+IL6) and in vivo experiments (serial transplantationin NOD/SCID mice) show that MPB CD34⁺ cells can be effectivelylong-term transduced by LV and maintain their proliferation,self renewal and multi-lineage differentiation potential. Weshow that expansion following transduction improves the engraftmentof transduced MPB CD34⁺ (4.6-fold expansion of SRC by limitingdilution studies). We propose ex-vivo expansion after transductionas an effective tool to improve gene therapy protocols withMPB.

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Authorcontributions: M.T.: Conception and design, Manuscript writing,animal models; L.G.: Conception and design, Manuscript writing,animal models; M.G.: Collection and/or assembly of data; V.L.:vector production; Y.P.: Molecular analysis; N.J.: Molecularanalysis; S.C.: in vitro experiments; C.C.: vector production;L.C.: Collection and/or assembly of data; G.M.: Data analysisand interpretation; F.F.: Financial support, Provision of studymaterial; A.T.: Provision of study material; M.A.: Financialsupport, Final approval of manuscript; W.P.: Conception anddesign, Financial support, Manuscript writing, Final approvalof manuscript.

M. Tesio and L. Gammaitoni contributed equallyto this work.
Key Words. Ex vivo gene transfer, hematopoietic stem cells (HSCs), enhanced green fluorescent protein (EGFP), expansion, mobilized peripheral blood