Evaluation of a nanoparticle-based gene therapy vector for treatment of Sanfilippo syndrome.

Author: William Scott

Scott, William, 2023 Evaluation of a nanoparticle-based gene therapy vector for treatment of Sanfilippo syndrome., Flinders University, College of Medicine and Public Health

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Mucopolysaccharidosis (MPS) IIIA, or Sanfilippo syndrome, is a lysosomal storage disorder of the mucopolysaccharidosis family, where a mutated N-sulfoglucosamine sulfohydrolase (SGSH) gene results in impaired production or function of the sulfamidase enzyme. This enzyme normally plays a critical role in the degradation of heparan sulfate, a long-chain glycosaminoglycan. As a result of this enzyme deficiency, heparan sulfate builds up in body tissues and causes a range of issues with an early onset of 2-4 years of age, most notably progressive and severe neurological impairment that eventually requires full-time care and leads to death by the late teens to early twenties.

Currently, no treatment exists for Sanfilippo syndrome, although multiple therapeutic approaches are being investigated. Importantly, removal of the accumulated excess heparan sulfate does not correct accumulated damage, so treatment must be started from an early age. One of the most promising avenues is gene therapy, where plasmids containing wildtype SGSH genes are delivered into cells via a vector to then express the required enzyme continuously in the body. Such therapies could provide a long-lasting treatment effect, although they would need to be repeated every few years as the number of cells in the patient’s body multiplies. Unfortunately, the currently used viral vectors face major issues with immunogenicity and pre-existing antibodies. This means that viral vectors are not suitable for a large portion of patients, and each successive treatment for a given patient will be less effective, rendering the approach unable to provide the repeated treatments needed over a patient’s lifetime.

Lipid nanoparticles are an emerging area of research that have the potential to provide alternative, non-immunogenic vector. Prior studies showed that a specific liposome formulation effectively induced expression of a green fluorescent protein in cell and mouse models. This project hypothesized that the nanoparticles would also effectively deliver the SGSH gene and effectively induce sulfamidase expression in similar models. To achieve this, a batch of liposome nanoparticles was created encapsulating a designed, SGSH-carrying pDNA plasmid, analysed to ensure purity and appropriate properties, and used to transfect wildtype and MPS-IIIA human fibroblasts across multiple experiments. The transfected cells were tested for heparan sulfate levels, lysosomal storage, and autophagic activity. Additionally, a second, fresh batch of nanoparticles were prepared, analysed, and delivered into wildtype and MPS-IIIA disease model mice via intercranial injection. These mice were later euthanised and tissues were collected. Brain samples were subsequently analysed for heparan sulfate levels and sulfamidase enzyme activity.

The results of the nanoparticle verification steps were positive, indicating an encapsulation ratio of approximately 80%, and a ~100-120nm size within the desired range indicted by the literature. However, the in vitro tests were only conducted for 4-day durations due to observed mortality in the initial experiment, and resulting in vitro data was inconclusive, with initial results not achieving statistical significance, and subsequent results indicating an increase in lysosomal storage and autophagic activity after treatment with the nanoparticle formulation.

This could be due to a mixture of variables potentially masking any treatment effects, such as lysosomal loading and induced autophagy due to cellular metabolism of lipid residues within the experimental timeframe, or oxidative stress affecting the outermost wells of treatment plates. Adjusted experimental designs with a focus on extended durations or randomisation could control for these factors, and modification of the liposome formulation could also be pursued to improve the encapsulation ratio further.

The in vivo experiments were conducted without issue, but failed to produce observable therapeutic effects or correction, indicating that further investigation into the stability, transfection efficiency, and expression of the encapsulated DNA nanoparticles may be required to determine the reason for the lack of effect.

Keywords: Biotechnology, Biotech, MPS, Sanfilippo, Sanfilippo Syndrome, MPS IIIA, Nanoparticle, Gene Therapy, Genetic Therapy, Nanoparticle Vector, SGSH, Fibroblast, Liposome, Sulfamidase, Heparan Sulfate

Subject: Medical Biotechnology thesis

Thesis type: Masters
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Professor Kim Hemsley