22 Insertional mutagenesis represents a major concern for gene therapy applications. Such a safety concern has dramatically risen after serious events in some of the patients with X-linked severe combined immunodeficiency, who received gene therapy treatment with murine oncoretroviral vectors.23,24 So far, in selleck Romidepsin contrast to oncoretroviral vectors, no adverse events have been reported after gene transfer with human immunodeficiency virus-1�Cderived lentiviral vector, and tumorigenesis was not stimulated in tumor-prone mice after human immunodeficiency virus-1�C derived lentiviral vector integration.25,26 However, preclinical animal and clinical studies may underscore vector genotoxicity because of the short animal lifespan as compared to humans, and because some observed adverse events might be due to negative synergy with underlying disease and other treatments.

Therefore, it is important to allow conditional ablation of transduced hepatocytes when required. The HSV-TK/GCV suicide strategy has been proven to be safe in a clinical trial of cancer gene therapy27 or modulation of graft-versus-host disease.28 The combined expression of the suicide HSV-TK gene with a therapeutic gene (in our study, the marker gene EPO) has, therefore, a clinically relevant application in liver gene therapy. HSV-TK�Cexpressing cells could be selectively eliminated by GCV treatment, a drug commonly used in clinical medicine for viral infections, which in itself is not toxic, but is converted to a toxic drug by viral thymidine kinase phosphorylation.

Cells expressing HSV-TK are producing highly toxic GCV-triphosphates that lead to cell death by inhibiting cellular DNA polymerase activity.29 Human, or simian in our study, thymidine kinase has narrow nucleotide specificity and is unable to activate the drug. We demonstrated HSV-TK functionality of the mTTR-cmEPO-TK vector both in human hepatic cell lines and in vivo in macaques upon GCV treatment. It should be noted that we waited for up to 16 months before complete ablation of transduced hepatocytes in macaques with GCV treatment, as assessed by qPCR. This data gave additional evidence that transduced hepatocytes were present and functional in the long term and that the mTTR promoter was not silenced.

This could be interpreted as an absence of immune response against the transgenes, but we did not perform assay to directly evaluate the presence of a cellular immune response against HSV-TK (or cmEPO), or this could be explained by a selection of low expressing EPO/HSV-TK hepatocytes over time, and these cells could be responsible for the residual EPO expression or HSV-TK in the long term. This finding demonstrates the possibility to selectively eliminate the transplanted cells, and suggest they could be similarly AV-951 killed if they displayed uncontrolled proliferation. Thus, the inclusion of the HSV-TK gene in the lentiviral vectors constitutes an additional guarantee for biosafety.