Sustained Correction of a Murine Model of Phenylketonuria following a Single Intravenous Administration of AAVHSC15-PAH

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 Pah enu2 mice. The AAVHSC15 vector containing a codon-optimized form of the human phenylalanine hydroxylase cDNA was administered as a single intravenous dose to Pah enu2 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 Pah enu2 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.

Development of an mRNA replacement therapy for phenylketonuria

“…The gender differences are consistent with what has been reported for Phe-lowering therapies using the PKU mice model, 13 , 14 , 15 , 16 , 17 which seems to be specific for the rodent model since it has not been reported in either non-human primates or in humans. 17 , 18 , 19

Figure 5 Plasma Phe pharmacodynamics following a repeating dosing of LUNAR-hPAH mRNA (LUNAR 1) (A–C) Pah enu2 homozygous mice maintained on a standard diet and water ad libitum were dosed five times with either vehicle (LUNAR buffer) or 3 mg/kg of LUNAR-hPAH mRNA at 3-day intervals. (A) Plasma Phe levels were quantified every 24 h in males (left graph, lines) and females (right graph, dotted line) in vehicle (blue lane) and treated (orange) groups.

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“…Recent advances in gene therapy have shown promising results in PKU mouse models with long-lasting reductions in Phe levels and partial liver correction. 14 , 15 , 16 , 17 Some of these therapeutic approaches are currently being tested in ongoing clinical trials with more efficient adeno-associated virus (AAV)-based delivery approaches to minimize effects associated with commonly known immune responses to the viral capsid, existing resistance to AAV, low transduction, or potential non-specific insertion. 28 The neonate's population is likely not suitable to PKU gene therapeutic approaches due to the wash-out of the transgene in hepatocytes in a still growing liver.…”

Natural variations in AAVHSC16 significantly reduce liver tropism and maintain broad distribution to periphery and CNS

“…We set out to identify the contribution of each unique residue on AAVHSC16, 501I or 706C, to lowered galactose binding by mutagenizing residues 501 and 706 on AAVHSC15, an AAVHSC that also contains 505R ( Table 1 , Table 2 and 2 ) and the most studied capsid in the AAVHSC panel. 8 , 27 , 28 , 29 As AAVHSC16 did not have robust binding to or transduction of Pro5 or Lec2 cells, we included additional mutant CHO cell lines to assess AAVHSC16 and mutagenized capsid binding to other terminal glycans ( Figures 1 A and 3 ). 24 , 30 , 31 The lack of terminal galactose was confirmed in Lec1 and Lec8 cells ( Figure S1 B).…”

Adeno-Associated Virus and Hematopoietic Stem Cells: The Potential of Adeno-Associated Virus Hematopoietic Stem Cells in Genetic Medicines

“…In 2019, Homology Medicines initiated a Phase1/2 open-label, randomized, concurrently controlled, dose escalation clinical gene therapy trial for PKU using an AAVHSC15 vector (pheNIX study, ClinicalTrials.gov Identifier NCT03952156). This trial, based upon Ahmed et al, 73 is designed to test the safety and efficacy of a single intravenous injection of an AAVHSC15 vector encoding a functional copy of the phenylalanine hydroxylase (PAH) gene in adults with PKU. PKU is an autosomal recessive inborn error of metabolism due to an absence of functional PAH, a hepatic enzyme that converts phenylalanine to tyrosine.…”