ORIGINAL ARTICLE Rhadinovirus vector-derived human telomerase reverse transcriptase expression in primary T cells T Toptan 1,2 , A Ensser 3 and H Fickenscher 1,2 1 Institute for Infection Medicine, Christian-Albrecht University of Kiel, Kiel, Germany; 2 Department of Infectious Diseases, Virology, Ruprecht-Karl University of Heidelberg, Heidelberg, Germany and 3 Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany The rhadinovirus herpesvirus saimiri (HVS) as a gene delivery vector allows large DNA insertions and long-termed gene expression. In the case of T-cell transduction, such vectors use the viral transformation-associated genes of HVS C488 for T-cell amplification. In this report, we investigated whether the gene for the catalytic telomerase subunit human telomerase reverse transcriptase (hTERT) can substitute for the transformation-associated genes in rhadinoviral T-cell transduction and amplification. By using virus mutants generated by en passant mutagenesis from bacterial artificial chromosomes, we observed a very early and functional transgene expression even by virus mutants without transformation-associated genes. The markers of T-cell transformation by HVS, namely CD2 hyperreactivity, overexpression of interleukin-26, and of the tyrosine kinase Lyn could neither be induced nor enhanced by ectopic hTERT expression. When the viral transformation-associated genes were replaced by the hTERT gene, it was not sufficient for growth transformation, although hTERT was efficiently transduced and functionally expressed by the rhadinovirus vector. Thus, the transformation-associated proteins StpC and Tip are responsible for the T-cell phenotype after transduction by HVS and, additionally, modulate telomerase activity independently of hTERT expression. Gene Therapy (2010) 17, 653–661; doi:10.1038/gt.2010.3; published online 18 February 2010 Keywords: rhadinovirus; herpesvirus saimiri; telomerase; hTERT; IL-26; CD2 Introduction T-cell-mediated adoptive immunotherapy is laborious because of several reasons, such as inefficient transduc- tion, suboptimal transgene expression or the limited life span of T lymphocytes in vitro. One explanation for the latter case may be the progressive telomere erosion by 50–200 bp in each cell division, leading to replicative senescence in somatic cells and T cells, unless telomerase is reexpressed. 1 Telomerase consists of an RNA template and a catalytic subunit, human telomerase reverse trans- criptase (hTERT). 2 It is naturally present in germ cells, fetal tissues, inflammatory cells and in most tumor cells, but not in somatic cells. Telomerase activity is induced in activated T cells in vivo, and can therefore sustain the telomere length and replication capacity during acute immune response and, thus, provide long-term memory. 3 hTERT is responsible for telomere maintenance in immune cells. 4 Ectopic hTERT expression in various human cell types including T lymphocytes reconstitutes telomerase activity, prevents telomere erosion and extends the life span of these cells without altering their functional and phenotypic properties. 5–10 Although anti- gen-specific T cells are rescued from apoptosis during primary activation by telomerase expression, repeated antigenic stimulation leads to telomere shortening due to the loss of telomerase inducibility. 11,12 T-cell memory erosion has an important role in chronic infections related to severe pathology, especially in elderly indivi- duals and in immunosuppressed patients. Hence, hTERT may be useful to circumvent the limitations of various therapeutic T-cell applications. Herpesvirus saimiri (HVS) belongs to the subfamily of g 2 -herpesviruses or rhadinoviruses. In contrast to the apathogenic persistence of HVS in its natural host, the squirrel monkey Saimiri sciureus, infected New World primates of other species develop fulminant lympho- proliferative disorders. HVS strains can be assigned to three subgroups, namely A, B and C, according to their oncogenic potential and their transformation-associated genes localized close to the left end of the genome. HVS C488 consists of an L-DNA coding for viral proteins which is flanked by noncoding repetitive sequences, termed ‘H-DNA’. 13–15 HVS strains of subgroup C such as C488 can transduce primary human T cells to stable proliferation, whereby the viral genomes are maintained as episomes in high copy numbers. 14,16 The transforma- tion-associated genes, tyrosine kinase-interacting protein (tip) and saimiri transformation-associated protein of subgroup C (stpC) are located at the left end of the L-DNA. 17,18 They are translated from a single bicistronic mRNA, and their transcription can be induced by T-cell activation. 19 Although these genes are dispensable for virus replication, their deletion abolishes the transforma- tion capacity of HVS C488. 20,21 Furthermore, growth- transformed human T cells do not release infectious Received 30 October 2009; revised 10 January 2010; accepted 12 January 2010; published online 18 February 2010 Correspondence: Dr H Fickenscher, Institute for Infection Medicine, Brunswiker Str. 4, 24105 Kiel, Germany. E-mail: fickenscher@infmed.uni-kiel.de Gene Therapy (2010) 17, 653–661 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt