http://www.loc.gov/mods/v3 http://www.loc.gov/standards/mods/v3/mods-3-2.xsd
Grisly World of MYOSIN-5A: Griscelli Syndrome Type 1 and the Search for an Effective Gene Therapy
Griscelli Syndrome type 1 (GS1) is an ultra-rare, autosomal recessive human disease that affects vesicle and membrane trafficking caused by a mutation in the Myosin-Va gene. Myosin-Va is an unconventional motor protein that is highly expressed in the nervous system and skin. The use of a GS1 rat model with a spontaneously occurring point mutation in the Myosin-Va gene, which leads to a loss of functional MYO5A, allows for an in-depth analysis of GS1 and the development of potential therapies. Tissue from wildtype, heterozygous, and mutant rats were analyzed to identify molecular changes and markers to evaluate GS1 and therapeutic efficacy. Protein, RNA, immunostaining, and weight gain analyses determined that GS1 rats have a significantly decreased growth curve compared to non-mutant littermates. Furthermore, by postnatal day 30 GS1 rats demonstrated differential protein expression related to oxidative stress and expressed multiple different Myo5a mRNA splicing isoforms compared to wildtype. Though more work is needed to categorize the molecular phenotype of the GS1 rat, the data provides a starting point for evaluating therapeutic effectiveness. Two gene therapies were also developed to address the current lack of treatment for GS1: a mutation-correcting gene editing therapy and a gene replacement therapy. The gene editing therapy utilized CRISPR/Cas9 and a DNA minicircle to correct the mutation through homology independent targeted integration. Primary GS1 cell cultures and PC-12 cells were treated with the CRISPR/Cas9 paradigm and proof-of-concept editing was demonstrated. The gene replacement therapy was delivered in an integrating lentiviral vector with myc-tagged, rat Myosin-Va mRNA expressed under the human synapsin 1 promoter. GS1 rat striatal slice cultures were given lentiviral or sham treatment and total RNA demonstrated a significant increase in the Myo5a-myc-tag expression in high dose lentiviral treated cultures compared to control, though neither protein expression nor immunostaining results produced an analogous result. Though further work is needed to identify GS1 molecular markers and to address whether either gene therapy will produce clinically effective results, the creation of these novel therapies provides a basis for improvement for what may one day be a treatment for GS1. The electronic version of this dissertation is accessible from https://hdl.handle.net/1969.1/197820
Doctor of Philosophy Texas A&M University 2022 https://hdl.handle.net/1969.1/197820