ABSTRACT
Adeno-associated viruses derived from human hematopoietic stem cells (AAVHSCs) are naturally occurring AAVs. Fifteen AAVHSCs have demonstrated broad biodistribution while displaying differences in transduction. We examine the structure-function relationships of these natural amino acid variations on cellular binding. We demonstrate that AAVHSC16 is the only AAVHSC that does not preferentially bind to terminal galactose. AAVHSC16 contains two unique amino acids, 501I and 706C, compared with other AAVHSCs. Through mutagenesis, we determined that residue 501 contributes to the lack of galactose binding. Structural analysis revealed that residue 501 is in proximity to the galactose binding pocket, hence confirming its functional role in galactose binding. Biodistribution analysis of AAVHSC16 indicated significantly less liver tropism in mice and non-human primates compared with other clade F members, likely associated with overall binding differences observed in vitro. AAVHSC16 maintained robust tropism to other key tissues in the peripheral and central nervous systems after intravenous injection, including to the brain, heart, and gastrocnemius. Importantly, AAVHSC16 did not induce elevated liver enzyme levels in non-human primates after intravenous injection at high doses. The unique glycan binding and tropism of AAVHSC16 makes this naturally occurring capsid an attractive candidate for therapies requiring less liver tropism while maintaining broad biodistribution.
ABSTRACT
Phenylketonuria is an inborn error of metabolism caused by loss of function of the liver-expressed enzyme phenylalanine hydroxylase and is characterized by elevated systemic phenylalanine levels that are neurotoxic. Current therapies do not address the underlying genetic disease or restore the natural metabolic pathway resulting in the conversion of phenylalanine to tyrosine. A family of hepatotropic clade F adeno-associated viruses (AAVs) was isolated from human CD34+ hematopoietic stem cells (HSCs) and one (AAVHSC15) was utilized to deliver a vector to correct the phenylketonuria phenotype in Pahenu2 mice. The AAVHSC15 vector containing a codon-optimized form of the human phenylalanine hydroxylase cDNA was administered as a single intravenous dose to Pahenu2 mice maintained on a phenylalanine-containing normal chow diet. Optimization of the transgene resulted in a vector that produced a sustained reduction in serum phenylalanine and normalized tyrosine levels for the lifespan of Pahenu2 mice. Brain levels of phenylalanine and the downstream serotonin metabolite 5-hydroxyindoleacetic acid were restored. In addition, the coat color of treated mice darkened following treatment, indicating restoration of the phenylalanine metabolic pathway. Taken together, these data support the potential of an AAVHSC15-based gene therapy as an investigational therapeutic for phenylketonuria patients.
