Directed protein evolution as a tool to innovate gene therapy for hereditary tyrosinemia type 1

Jessie Neuckermans, Haaike Colemonts-Vroninks, Lionel Marcélis, Alan Mertens, Ulrich Schwaneberg, Joery De Kock

Onderzoeksoutput: Poster


Background and Aims: Hereditary tyrosinemia type 1 (HT1) is a life-threatening hepatorenal disorder characterised by a dysfunctional fumarylacetoacetase (FAH) enzyme. HT1 can be effectively treated with the drug nitisinone (NTBC), a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPD), an enzyme upstream of FAH. Gene therapy with the wildtype version of FAH would require withdrawal of NTBC due to the upstream metabolic block. Nonetheless, a low transduction efficiency impedes normalisation of the patient’s metabolome and due to the cell-autonomous pathophysiology of the disease, withdrawal of NTBC is unacceptable. As a first step to solve the foregoing problem, we aimed to engineer an HPD enzyme with a significantly decreased sensitivity towards NTBC (HPD^NTBC) by the use of directed protein evolution technology.

Method: First, we developed a colorimetric bacterial whole-cell screening system that allows the evaluation of HPD variants in a high-throughput and a robust fashion (Z’ = 0.93 ± 0.02). The principle of the screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. Next, the KnowVolution approach was used to engineer a human HPD enzyme with a significantly decreased sensitivity towards NTBC. This comprised the identification and determination of beneficial amino acid positions, substitutions and their recombination. Technologies that were used in iterative rounds of evolution included error prone (ep) PCR, site saturation mutagenesis (SSM) and site-directed mutagenesis (SDM).

Results: EpPCR allowed to identify 12 amino acid substitutions that decreased the sensitivity of human HPD towards NTBC. Combination of 8 out of 12 individual beneficial amino acid substitutions by SSM and SDM into a single lead variant further decreased the sensitivity of the engineered human HPD^NTBC enzyme towards NTBC as well as other triketone inhibitors. More specifically, the engineered lead variant exhibited significantly increased IC50 values versus wild type human HPD for the triketone inhibitors nitisinone (20.2-fold), mesotrione (14.1-fold), sulcotrione (19.8-fold) and tembotrione (9.0-fold) (Fig).

Conclusion: This NTBC insensitive human HPD variant comprises the first step in the development of new gene therapeutic approaches for the treatment of HT1 that are functional under NTBC treatment.
Originele taal-2English
StatusPublished - mrt 2020
EvenementEASL 2020: The International Liver Congress - London, United Kingdom
Duur: 15 apr 202019 apr 2020


ConferenceEASL 2020
LandUnited Kingdom


  • EASL
  • ICL

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