Date of Award
College of Pharmacy
Director of Thesis
Michael Wyatt, PhD
Douglas Pittman, PhD
Phenylketonuria, now known as phenylalanine hydroxylase (PAH) deficiency, is a genetic disorder of metabolism affecting approximately one in every 15,000 infants born in the United States. Patients have nonfunctional PAH enzyme secondary to one or more genetic mutations. The enzyme deficit results in destructive supraphysiologic blood phenylalanine levels upon consumption of the essential dietary amino acid phenylalanine. Current standards of care mitigate signs and symptoms of the disorder, but do not approach a cure. The methods for creating a prototype pharmabiotic as an innovative treatment strategy for PAH deficiency are described herein.
DNA molecular cloning techniques were utilized to engineer a novel plasmid termed LiLi5, which was subsequently transformed into Lactobacillus helveticus. A cDNA for human PAH was amplified and inserted into a gram-neutral shuttle vector to create LiLi5, the bacterial plasmid which induces the expression of human phenylalanine hydroxylase. The LiLi5 plasmid was confirmed by restriction enzyme digest and sequencing of the cDNA insert. Successful transformation of LiLi5 into Lactobacillus helveticus generated a novel strain of Lactobacillus, which was christened “HELin”. PCR-based detection confirmed the presence of the human PAH cDNA in HELin.
Phenylalanine hydroxylase deficiency is a debilitating disorder causing somatic, psychosocial, and financial distress for patients and their families. Current treatment strategies do not adequately address the decreased quality of life experienced by the associated patient population. Lactobacillus helveticus is a GRAS gram-positive bacterial species commonly formulated in commercially marketed probiotics. The novel strain of Lactobacillus expressing human phenylalanine hydroxylase that has been named ‘HELin’ is proposed to be formulated into a pharmabiotic for testing as a new treatment for phenylalanine hydroxylase deficiency. Patent pending.
LeBegue, Chloé Elizabeth, "The Pharmabiotic for Phenylketonuria: Development of a Novel Therapeutic" (2019). Senior Theses. 267.
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