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Supervisor: Stavros Loukogeorgakis, Alessia Cavazza, Dominic Scaglioni
Project Description:
Background:
Alpha thalassemia is one of the most common hemoglobinopathies worldwide, caused by mutations in the HBA1 and HBA2 genes. Patients with severe disease form are at high risk of fetal demise without in-utero intervention. Gene therapy provides an attractive method for the curative treatment of the severe form the disease in which no functional HBA1 or HBA2 genes are present. However, efficient targeting and correction of hematopoietic stem cells (HSCs) in vivo has proven challenging.
Aims/Objectives
This project will explore prenatal gene therapy, looking at both viral and non-viral vectors. Virus-like particles (VLPs) are self-assembling protein nanoparticles that have great promise as vectors to deliver gene editing agents in vivo. VLPs are remarkably efficient for entry into human T, B, and HSCs. We will use VLPs to specifically target HSCs and perform in-utero gene addition as a potential curative treatment for the severe form of alpha-thalassemia. Additionally, comparisons between viral (VLP) and non-viral (lipid nanoparticles) vectors for gene addition will be performed to develop an optimum in utero delivery strategy for gene editing components.
Methods
VLPs will be produced and optimised for in vitro gene addition. The VLPs will contain a genomic cleaving agent that consists of a Gag viral protein that is fused to a Cas9 alongside gRNA and donor DNA for gene addition. The VLP will also be pseudo-typed with a baboon endogenous virus envelope to enhance vector tropism for HSCs. The VLPs will subsequently be tested on primary cells in vitro and on a mouse alpha-thalassemia model in vivo. The project will include skills such as; cell culture (primary mammalian and producer cell line), VLP Manufacture and Transduction (plasmid transfections, VLP harvest), alongside animal handling.
Timeline
Initial months will be spent optimising the production of VLPs and pseudotyping for specific targeting of HSCs. In vitro studies on primary HSCs will then be performed to assess transfection and gene addition efficiency. Following initial production and characterisation, VLP experiments in a mouse model of alpha-thalassemia will be performed to better optimise VLP-mediated CRISPR/Cas9 gene addition in vivo. Alongside this, multilamellar lipid vesicles (MLVs) will also be produced and assessed as carrier vehicles for the delivery of CRISPR/Cas9 gene addition components. Comparisons between VLPs and MLVs will be performed, both in vitro and in vivo, to determine the optimal strategy for the targeted delivery of gene addition components in utero.
Relevant Publications
1. Loukogeorgakis, S. P. & Flake, A. W. In utero stem cell and gene therapy: current status and future perspectives. Eur J Pediatr Surg 24, 237–245 (2014).
2. Gutierrez-Guerrero, A. et al. Baboon Envelope Pseudotyped “Nanoblades” Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells. Front. Genome Ed. 3, (2021).
3. Banskota, S. et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250-265.e16 (2022).
Contact Information:
Stavros Loukogeorgakis