State-of-the-art 2023 on gene therapy for phenylketonuria

Abstract

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.

Keywords: adeno-associated virus; gene editing; gene therapy; mRNA therapy; phenylalanine; phenylalanine hydroxylase; phenylketonuria.

Figure 1

Phenylketonuria (PKU) is caused by recessively-inherited variants in the phenylalanine hydroxylase (PAH) gene (Panel A). Phenylalanine hydroxylase (PAH) is a homotetramer that catalyzes the irreversible conversion of phenylalanine (Phe) to tyrosine (Tyr). The reaction requires reduced tetrahydrobiopterin (BH₄), iron, and molecular oxygen as cofactors (not shown). In the absence of PAH activity, phenylalanine accumulates in tissues and is non-enzymatically deaminated to phenylpyruvate and further oxidized to other phenylketones, leading to the eponymic name phenylketonuria (PKU). Biallelic PAH variants encode variant PAH messenger RNA (mRNA) which then lead to either unstable, poorly active, or inactive PAH protein and impaired ability to hydroxylate Phe to Tyr in liver. Genetic therapies (Panel B) aim to restore liver PAH expression by gene addition, or CRISPR/Cas-based gene or base editing; i.e. several different treatment approaches to accomplish this goal are under preclinical investigation with mice, including (1) gene addition, (2) delivery of therapeutic mRNA via lipid nanoparticles (LNP), (3) gene editing/correction, or (4) gene insertion. Gene addition is currently most frequently attempted through delivery of a PAH expression cassette to hepatocytes using either recombinant adeno-associated virus (rAAV) vectors or non-viral (minicircle) vectors. rAAV genomes penetrate into the hepatocyte nucleus and predominantly remain episomal, not interacting with the host genome, but expressing the therapeutic transgene. Several different gene or base editing technologies are available to accomplish site-specific correction of the pathologic variant back to the wild type sequence in gene correction. Some of these editing methods suffer from low correction frequency; all must be redesigned for every specific pathologic variant to be targeted. Gene insertion yields a combination of gene addition and gene correction by permanently inserting an entire PAH expression cassette somewhere into the hepatocyte genome.