Developing RNA sequences to regulate therapeutic genes expression

  1. ROVIRA BARREIRA, ERIC
Supervised by:
  1. Fernando Pastor Rodríguez Director
  2. Maria Purificacion Fortes Alonso Director

Defence university: Universidad de Navarra

Fecha de defensa: 06 October 2022

Committee:
  1. Alfredo Berzal Herranz Chair
  2. Cristian Smerdou Picazo Secretary
  3. Lorea Blazquez Garcia Committee member
  4. Andoni Garitano Trojaola Committee member
  5. Beatrix Suess Committee member
Department:
  1. (FM) Hematología

Type: Thesis

Teseo: 756213 DIALNET

Abstract

In our lab we believe that by studying natural gene-expression regulatory mechanisms, we can acquire the necessary knowledge to engineer novel regulatory systems. In this thesis we decided to focus on a master regulator of gene expression in order to develop 2 novel RNA-based gene regulatory platforms. As a first approach, we used RNA sequences to recruit a positive regulator of gene expression. In this work, we demonstrate that contrary to what had been described so far, the U1 snRNP-specific protein U1A acts as a positive regulator of many cellular genes, including crucial regulators of cell proliferation and migration. In addition, we show that targeting U1A with RNAi leads to reduced proliferative capacity and migration potential of hepatocellular carcinoma cells. Finally, we show that by transferring endogenous U1A-binding motifs to exogenous genes we are able to upregulate their expression in different cell lines in both transient and stable systems. As an alternative RNA-based regulatory system, we employ riboswitches to modulate the accessibility of binding sites of a master regulator complex. In this system we demonstrated that we are able to modulate the expression of both cytoplasmic and membrane reporter genes. In addition, we describe a novel riboswitch high-throughput engineering platform, which allowed us to isolate novel riboswitches with enhanced binding capacity to such gene master regulator in the absence of inducer drug. This translated into reduced leakage of our system. Moreover, we combined multiple single riboswitches generating constructs which are possess a dynamic range of over 30-fold. Finally, we provided evidence that our system has the potential to work in vivo. CAR T cells represent a potent antitumoral strategy, which is based on the genetic modification of human T lymphocytes in order for them to express a chimeric antigen receptor (CAR), which directs their activity against tumor-specific/tumor-associated antigens. Despite their great success, their potent activity results in many cases in a severe multisystemic inflammatory syndrome, which may be lethal in some individuals. Bearing in mind the clear need for novel regulatory systems to modulate CAR expression, we identified several drugs with clinically appealing properties in order to develop novel riboswitches which induce gene expression in their presence. Provided there were no reported riboswitches which can recognize these novel ligands, we developed yet another new high-throughput technique to identify functional riboswitches. In addition, we show we are able to regulate EGFP expression in mammalian cells. Finally, we engineered CD19 CAR T cells with some of such new riboswitches and showed that in the absence of the drug these cells present reduced proliferation and IFN gamma secretion capacities. Moreover, addition of the riboswitch ligand restores their functionality to a level equivalent to a conventional CD19-CAR.