Volker Patzel
Department of Microbiology & Immunology, Singapore
Title: Selective killing of virus-transduced or cancer cells using a RNA trans-splicing based suicide gene therapy approach
Biography
Biography: Volker Patzel
Abstract
Statement of the Problem: Acquired and inherited genetic disorders are characterized by the cellular expression of aberrant transcripts and proteins. Viruses such as the human papillomaviruses (HPV) or the human immunodeficiency virus type 1 (HIV-1) integrate their DNA into the genome of the infected host cell and there is no way to get rid of the viral DNA anymore. Similarly, many cancers are characterized by oncogene expression. Methodology & Theoretical Orientation: We explored a Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy approach for selective killing of virus-transduced or cancer cells. First we studied the highly efficient mechanism of trans-splicing among transcripts of the simian virus 40 (SV40). Then we employed molecular features of SV40 RNA trans-splicing and computational RNA structure design to improve both on-target activity and specificity of the trans-splicing RNA (tsRNA). As molecular targets we selected the a-fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC), human papillomavirus type 16 (HPV-16), or human immunodeficiency virus type 1 (HIV-1) pre-mRNA. Findings: While unstructured mismatched target binding domains significantly improved 3’ exon replacement, 5’ exon replacement correlated with the thermodynamic stability of the tsRNA 3’ end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA harbouring multiple target binding domains shielding alternative on-target and blinding non-target splicing events. Rationally designed tsRNAs efficiently and selectively triggered death of HPV-16, HIV-1 or AFP-positive cells. Dual-targeting tsRNA simultaneously targeting AFP and a second HCC biomarker triggered enhanced cell death at 10-fold lower GCV doses. Conclusion & Significance: Our observations suggest RNA trans-splicing represents a promising approach to suicide gene therapy targeting viral infection, cancer or other diseases characterized by the expression of disease-specific pre-mRNA biomarkers