title: Reprogramming human cells using SMAR vectors  and  Potentiation of H-1PV oncolysis by Ledipasvir
creator: Hartley, Anna
subject: ddc-500
subject: 500 Natural sciences and mathematics
description: This two-part thesis explores the utility of non-viral DNA vectors for reprogramming human cells, as well as the potentiation of the oncolytic virus H-1 parvovirus (H-1PV) by a novel combination with the antiviral drug Ledipasvir.    Pluripotent stem cells are an attractive tool for regenerative medicine due to their capacity for unlimited self-renewal and their ability to differentiate into any somatic cell type. However, the generation of pluripotent stem cells from human embryos raises ethical challenges and is strictly regulated as a result. The advent of cellular reprogramming has now made it possible to generate induced pluripotent stem cells (iPSCs) from a patient’s own cells, or for derivation of off-the-shelf cell therapies without the ethical concerns of embryonic stem cells. Nevertheless, current methods for the generation of iPSCs for clinical application cause concerns due to the use of viruses and viral components. Lentiviral transduction is a popular method to introduce reprogramming factors into cells, however they carry with them a high risk of insertional mutagenesis. iPSCs which are currently employed in a therapeutic manner in clinical trials are exclusively derived using the oriP/EBNA-1 vector system, which makes use of the Epstein Barr Virus Nuclear Antigen 1, a protein which has been implicated in oncogenesis, and is known to induce immune activation in iPSCs. Thus, there is a clear need to improve the safety of the reprogramming process to generate iPSCs with the least possible damage and immune activation.   In this thesis, I show for the first time that DNA vectors which comprise Scaffold/Matrix Attachment Regions (S/MARs) can be used to reprogram healthy neonatal human dermal fibroblasts, eliminating the need for viral components. SMAR vectors are retained episomally in cells without integration, but require only a single transfection for successful reprogramming, a feature which is highly attractive for current good manufacturing practice (cGMP). SMAR iPSCs can be generated at practical efficiencies and are phenotypically indistinguishable from traditional EBNA iPSCs. They also show strong similarity to human embryonic stem cell lines in their gene expression. Importantly, SMAR iPSCs show differentiation capacity into cell of all three embryonic lineages, confirming their pluripotency. Surprisingly, modifications to the vectors to reduce their immunogenicity and improve their retention in cells proved counterproductive, as these optimised nanoSMAR vectors were unable to fully reprogram cells. The genetic stability of SMAR iPSCs is comparable to that of EBNA iPSCs, and they show the potential for loss of the vector after reprogramming to generate vector free iPSCs. Further fine-tuning of the SMAR vector system to ensure passive vector loss after reprogramming is discussed. This work thus represents a step towards virus free, vector free and factor free reprogramming for clinical applications.    The field of oncolytic virotherapy has enjoyed increasing clinical success in the past decade, with the worldwide approval of four oncolytic viruses to date. One of the oncolytic viruses under clinical evaluation is H-1PV, a rodent protoparvovirus which is non-pathogenic to humans and possesses natural oncolytic activity. Its replication is strictly dependent on a transformed phenotype in human cells, giving it a good safety profile for clinical use. This virus has shown considerable promise in early-stage clinical trials for the treatment of both high-grade glioma and pancreatic ductal adenocarcinoma (PDAC), two cancer modalities for which treatment options remain limited and prognosis is dismal. H-1PV treatment in these settings was safe and well tolerated, with some early signs of efficacy including evidence of viral replication in the tumour bed and immune conversion of the tumour microenvironment, as well as an improvement in patient survival in comparison to historical controls. Nonetheless, H-1PV monotherapy was not curative under the regimes used, highlighting a need for the improvement of oncolytic H-1PV therapy.  One approach for this with rapid translational potential is the combination therapy of H-1PV with other drugs to improve its oncolytic activity. This approach has already shown promise in the successful potentiation of H-1PV in combination with the histone deacetylase inhibitor valproic acid and pro-apoptotic BH3 mimetics, such as ABT-737. Here I present a high-throughput screening approach which identified the FDA-approved antiviral drug Ledipasvir as a potentiator of H-1PV oncolysis in human cells. Importantly, Ledipasvir co-treatment improves the oncolytic capabilities of H-1PV in a wide range of cancer cell lines spanning diverse tumour types, including primary patient-derived PDAC cultures. I determined that the cell death induced by the Ledipasvir-H-1PV combination is at least in part apoptotic, but the drug is unable to improve viral replication. Instead, Ledipasvir improves the oncotoxic properties of the major effector protein NS1, albeit without directly binding to NS1. A series of unbiased screening approaches revealed a complex interplay between Ledipasvir, H-1PV, and the pro-survival PI3K/Akt/mTOR signalling pathway. While H-1PV alone activates Akt signalling, this becomes hyperactivated upon the addition of Ledipasvir, and may serve to improve the functionality of NS1. Further downstream, inhibition of mTOR by the small molecule Torin was able to abrogate the combinatorial effects of H-1PV and Ledipasvir, without affecting oncolysis by H-1PV alone. I propose a tentative model in which the combination of Ledipasvir and H-1PV modulate components of the PI3K/Akt/mTOR pathway to improve the functionality and oncotoxic capabilities of the H-1PV NS1 protein, ultimately improving viral oncolysis.
date: 2025
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/34990/1/PhD_Thesis_Anna_Hartley.pdf
identifier: DOI:10.11588/heidok.00034990
identifier: urn:nbn:de:bsz:16-heidok-349900
identifier:   Hartley, Anna  (2025) Reprogramming human cells using SMAR vectors and Potentiation of H-1PV oncolysis by Ledipasvir.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/34990/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng