Phenylketonuria (PKU) is considered to be a paradigm for a monogenic metabolic disorder but was never thought to be a primary application for human gene therapy due to established alternative treatment. However, somewhat unanticipated improvement in neuropsychiatric outcome upon long-term treatment of adults with PKU with enzyme substitution therapy might slowly change this assumption. In parallel, PKU was for a long time considered to be an excellent test system for experimental gene therapy of a Mendelian autosomal recessive defect of the liver due to an outstanding mouse model and the easy to analyze and well-defined therapeutic end point, that is, blood l-phenylalanine concentration. Lifelong treatment by targeting the mouse liver (or skeletal muscle) was achieved using different approaches, including (1) recombinant adeno-associated viral (rAAV) or nonviral naked DNA vector-based gene addition, (2) genome editing using base editors delivered by rAAV vectors, and (3) by delivering rAAVs for promoter-less insertion of the PAH-cDNA into the Pah locus. In this article we summarize the gene therapeutic attempts of correcting a mouse model for PKU and discuss the future implications for human gene therapy.
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References
1.
DonlonJ, SarkissianC, LevyH, et al. In: BeaudetAL, VogelsteinB, KinzlerKW, et al., eds. The Online Metabolic and Molecular Bases of Inherited Disease. New York, NY: The McGraw-Hill Companies, Inc., 2014.
2.
BlauN, van SpronsenFJ, LevyHL. Phenylketonuria. Lancet, 2010; 376:1417–1427.
BlauN, MartinezA, HoffmannGF, et al.DNAJC12 deficiency: a new strategy in the diagnosis of hyperphenylalaninemias. Mol Genet Metab, 2018; 123:1–5.
5.
GuthrieR, SusiA. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics, 1963; 32:338–343.
6.
LevyHL, SarkissianCN, ScriverCR. Phenylalanine ammonia lyase (PAL): from discovery to enzyme substitution therapy for phenylketonuria. Mol Genet Metab, 2018:124:223–229.
7.
VockleyJ, AnderssonHC, AntshelKM, et al.Phenylalanine hydroxylase deficiency: diagnosis and management guideline. Genet Med, 2014; 16:188–200.
8.
van SpronsenFJ, van WegbergAM, AhringK, et al.Key European guidelines for the diagnosis and management of patients with phenylketonuria. Lancet Diabetes Endocrinol, 2017; 5:743–756.
9.
van WegbergAMJ, MacDonaldA, AhringK, et al.The complete European guidelines on phenylketonuria: diagnosis and treatment. Orphanet J Rare Dis, 2017; 12:162.
10.
BilderDA, NoelJK, BakerER, et al.Systematic review and meta-analysis of neuropsychiatric symptoms and executive functioning in adults with phenylketonuria. Dev Neuropsychol, 2016; 41:245–260.
11.
DeRocheK, WelshM. Twenty-five years of research on neurocognitive outcomes in early-treated phenylketonuria: intelligence and executive function. Dev Neuropsychol, 2008; 33:474–504.
12.
ChristSE, HuijbregtsSC, de SonnevilleLM, et al.Executive function in early-treated phenylketonuria: profile and underlying mechanisms. Mol Genet Metab, 2010; 99:S22–S32.
13.
JahjaR, van SpronsenFJ, de SonnevilleLMJ, et al.Social-cognitive functioning and social skills in patients with early treated phenylketonuria: a PKU-COBESO study. J Inherit Metab Dis, 2016; 39:355–362.
14.
KochR, BurtonB, HogansonG, et al.Phenylketonuria in adulthood: a collaborative study. J Inherit Metab Dis, 2002; 25:333–346.
15.
LenkeRR, LevyHL. Maternal phenylketonuria and hyperphenylalaninemia. An international survey of the outcome of untreated and treated pregnancies. N Engl J Med, 1980; 303:1202–1208.
16.
LongoN, DimmockD, LevyH, et al.Evidence- and consensus-based recommendations for the use of pegvaliase in adults with phenylketonuria. Genet Med, 2018 [Epub ahead of print]; DOI: 10.1038/s41436-018-0403-z.
17.
MahanKC, GandhiMA, AnandS. Pegvaliase: a novel treatment option for adults with phenylketonuria. Curr Med Res Opin, 2019; 35:647–651.
18.
ThomasJ, LevyH, AmatoS, et al.Pegvaliase for the treatment of phenylketonuria: results of a long-term phase 3 clinical trial program (PRISM). Mol Genet Metab, 2018; 124:27–38.
19.
GuptaS, LauK, HardingCO, et al.Association of immune response with efficacy and safety outcomes in adults with phenylketonuria administered pegvaliase in phase 3 clinical trials. EBioMedicine, 2018; 37:366–373.
20.
PascucciT, RossiL, ColamartinoM, et al.A new therapy prevents intellectual disability in mouse with phenylketonuria. Mol Genet Metab, 2018; 124:39–49.
21.
IsabellaVM, HaBN, CastilloMJ, et al.Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol, 2018; 36:857–864.
22.
McDonaldJD, BodeVC, DoveWF, et al.Pahhph-5: a mouse mutant deficient in phenylalanine hydroxylase. Proc Natl Acad Sci U S A, 1990; 87:1965–1967.
23.
McDonaldJD, CharltonCK. Characterization of mutations at the mouse phenylalanine hydroxylase locus. Genomics, 1997; 39:402–405.
24.
HeintzC, TroxlerH, MartinezA, et al.Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry. Mol Genet Metab, 2012; 105:559–565.
25.
McDonaldJD. Postnatal growth in a mouse genetic model of classical phenylketonuria. Contemp Top Lab Anim Sci, 2000; 39:54–56.
26.
ZagredaL, GoodmanJ, DruinDP, et al.Cognitive deficits in a genetic mouse model of the most common biochemical cause of human mental retardation. J Neurosci, 1999; 19:6175–6182.
27.
WinnSR, SchererT, ThonyB, et al.Blood phenylalanine reduction corrects CNS dopamine and serotonin deficiencies and partially improves behavioral performance in adult phenylketonuric mice. Mol Genet Metab, 2018; 123:6–20.
28.
DobrowolskiSF, TourkovaIL, RobinsonLJ, et al.A bone mineralization defect in the Pah(enu2) model of classical phenylketonuria involves compromised mesenchymal stem cell differentiation. Mol Genet Metab, 2018; 125:193–199.
29.
Perez-DuenasB, CambraFJ, VilasecaMA, et al.New approach to osteopenia in phenylketonuric patients. Acta Paediatr, 2002; 91:899–904.
30.
van SpronsenFJ, BurgardP. The truth of treating patients with phenylketonuria after childhood: the need for a new guideline. J Inherit Metab Dis, 2008; 31:673–679.
31.
ChoS, McDonaldJD. Effect of maternal blood phenylalanine level on mouse maternal phenylketonuria offspring. Mol Genet Metab, 2001; 74:420–425.
32.
McDonaldJD, DyerCA, GailisL, et al.Cardiovascular defects among the progeny of mouse phenylketonuria females. Pediatr Res, 1997; 42:103–107.
HardingCO, DingZ, ThönyB. Gene and cell therapies for phenylketonuria (PKU). In: BlauN, ed. PKU and BH4: Advances in Phenylketonuria and Tetrahydrobiopterin. Heilbronn: SPS Publications, 2006.
36.
SarkissianCN, GamezA, ScriverCR. What we know that could influence future treatment of phenylketonuria. J Inherit Metab Dis, 2009; 32:3–9.
37.
ThonyB. Long-term correction of murine phenylketonuria by viral gene transfer: liver versus muscle. J Inherit Metab Dis, 2010; 33:677–680.
38.
Al HafidN, ChristodoulouJ. Phenylketonuria: a review of current and future treatments. Transl Pediatr, 2015; 4:304–317.
39.
HammanK, ClarkH, MontiniE, et al.Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria. Mol Ther, 2005; 12:337–344.
40.
HammanKJ, WinnSR, HardingCO. Hepatocytes from wild-type or heterozygous donors are equally effective in achieving successful therapeutic liver repopulation in murine phenylketonuria (PKU). Mol Genet Metab, 2011; 104:235–240.
41.
LinCM, TanY, LeeYM, et al.Expression of human phenylalanine hydroxylase activity in T lymphocytes of classical phenylketonuria children by retroviral-mediated gene transfer. J Inherit Metab Dis, 1997; 20:742–754.
42.
FangB, EisensmithRC, LiXH, et al.Gene therapy for phenylketonuria: phenotypic correction in a genetically deficient mouse model by adenovirus-mediated hepatic gene transfer. Gene Ther, 1994; 1:247–254.
43.
NagasakiY, MatsubaraY, TakanoH, et al.Reversal of hypopigmentation in phenylketonuria mice by adenovirus-mediated gene transfer. Pediatr Res, 1999; 45:465–473.
44.
OhHJ, ParkES, KangS, et al.Long-term enzymatic and phenotypic correction in the phenylketonuria mouse model by adeno-associated virus vector-mediated gene transfer. Pediatr Res, 2004; 56:278–284.
45.
MochizukiS, MizukamiH, OguraT, et al.Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice. Gene Ther, 2004; 11:1081–1086.
46.
EmburyJ, FrostS, CharronCE, et al.Hepatitis virus protein X-phenylalanine hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors. Gene Ther Mol Biol, 2008; 12:69–76.
47.
GaoGP, AlviraMR, WangL, et al.Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A, 2002; 99:11854–11859.
48.
HardingCO, GillinghamMB, HammanK, et al.Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria. Gene Ther, 2006; 13:457–462.
49.
DingZ, GeorgievP, ThonyB. Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer. Gene Ther, 2006; 13:587–593.
50.
YagiH, OguraT, MizukamiH, et al.Complete restoration of phenylalanine oxidation in phenylketonuria mouse by a self-complementary adeno-associated virus vector. J Gene Med, 2011; 13:114–122.
51.
ThonyB, DingZ, RebuffatA, et al.Phenotypic reversion of fair hair upon gene therapy of the phenylketonuria mice. Hum Gene Ther, 2014; 25; 573–574.
52.
RebuffatA, HardingCO, DingZ, et al.Comparison of adeno-associated virus pseudotype 1, 2, and 8 vectors administered by intramuscular injection in the treatment of murine phenylketonuria. Hum Gene Ther, 2010; 21:463–477.
53.
YagiH, SanechikaS, IchinoseH, et al.Recovery of neurogenic amines in phenylketonuria mice after liver-targeted gene therapy. Neuroreport, 2012; 23:30–34.
54.
WrightJ, EllsworthJF, St. Martin T, et al. Nuclease-free and promoter-less AAVHSC-mediated genome editing in vivo corrects the disease phenotype in a mouse model of phenylketonuria. Mol Ther, 2019; 27:462.
55.
DingZ, HardingCO, RebuffatA, et al.Correction of murine PKU following AAV-mediated intramuscular expression of a complete phenylalanine hydroxylating system. Mol Ther, 2008; 16:673–681.
56.
Al ShaerD, Al MusaimiO, AlbericioF, et al.2018 FDA tides harvest. Pharmaceuticals (Basel), 2019; 12:E52.
57.
ViecelliHM, HarbottleRP, WongSP, et al.Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver. Hepatology, 2014; 60:1035–1043.
58.
GasparV, de Melo-DiogoD, CostaE, et al.Minicircle DNA vectors for gene therapy: advances and applications. Expert Opin Biol Ther, 2015; 15:353–379.
59.
GillDR, PringleIA, HydeSC. Progress and prospects: the design and production of plasmid vectors. Gene Ther, 2009; 16:165–171.
60.
HydeSC, PringleIA, AbdullahS, et al.CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nat Biotechnol, 2008; 26:549–551.
61.
NairN, RinconMY, EvensH, et al.Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy. Blood, 2014; 123:3195–3199.
62.
Grisch-ChanHM, SchlegelA, SchererT, et al.Low-dose gene therapy for murine PKU using episomal naked DNA vectors expressing PAH from its endogenous liver promoter. Mol Ther Nucleic Acids, 2017; 7:339–349.
CongL, RanFA, CoxD, et al.Multiplex genome engineering using CRISPR/Cas systems. Science, 2013; 339:819–823.
65.
MaliP, YangL, EsveltKM, et al.RNA-guided human genome engineering via Cas9. Science, 2013; 339:823–826.
66.
BarnesDE. Non-homologous end joining as a mechanism of DNA repair. Curr Biol, 2001; 11:R455–R457.
67.
van den BoschM, LohmanPH, PastinkA. DNA double-strand break repair by homologous recombination. Biol Chem, 2002; 383:873–892.
68.
HuaiC, JiaC, SunR, et al.CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice. Hum Genet, 2017; 136:875–883.
69.
YinH, XueW, ChenS, et al.Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol, 2014; 32:551–553.
70.
OhmoriT, NagaoY, MizukamiH, et al.CRISPR/Cas9-mediated genome editing via postnatal administration of AAV vector cures haemophilia B mice. Sci Rep, 2017; 7:4159.
71.
AdikusumaF, PiltzS, CorbettMA, et al.Large deletions induced by Cas9 cleavage. Nature, 2018; 560:E8–E9.
72.
KosickiM, TombergK, BradleyA. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol, 2018; 36:765–771.
73.
HaapaniemiE, BotlaS, PerssonJ, et al.CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med, 2018; 24:927–930.
74.
IhryRJ, WorringerKA, SalickMR, et al.p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med, 2018; 24:939–946.
75.
GaudelliNM, KomorAC, ReesHA, et al.Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature, 2017; 551:464–471.
76.
KomorAC, KimYB, PackerMS, et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016; 533:420–424.
77.
ReesHA, KomorAC, YehWH, et al.Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun, 2017; 8:15790.
78.
SongC-Q, JiangT, RichterM, et al.Adenine base editing in an adult mouse model of tyrosinaemia. Nat Biomed Eng, 2019; DOI: 10.1038/s41551-019-0357-8.
79.
YehWH, ChiangH, ReesHA, et al.In vivo base editing of post-mitotic sensory cells. Nat Commun, 2018; 9:2184.
80.
VilligerL, Grisch-ChanHM, LindsayH, et al.Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat Med, 2018; 24:1519–1525.
81.
GrunewaldJ, ZhouR, GarciaSP, et al.Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature, 2019; 569; DOI: 10.1038/s41586-019-1161-z.
82.
JinS, ZongY, GaoQ, et al.Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science, 2019; 364:292–295.
83.
ZuoE, SunY, WeiW, et al.Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science, 2019; 364:289–292.
84.
WrightTL, EllsworthJF, AhmedSS, et al.A novel AAVHSC15 encoding human phenylalanine hydroxylase, in cynomolgus monkeys. Mol Ther, 2019; 27:67.
85.
CunninghamSC, SpinoulasA, CarpenterKH, et al.AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spfash mice. Mol Ther, 2009; 17:1340–1346.
86.
WangL, WangH, BellP, et al.Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector. Hum Gene Ther, 2012; 23:533–539.
87.
WangL, BellP, LinJ, et al.AAV8-mediated hepatic gene transfer in infant rhesus monkeys (Macaca mulatta). Mol Ther, 2011; 19:2012–2020.
88.
BrownCS. Family reflections on phenylketonuria. Pediatr Res, 2018; 84:797–798.
89.
MilaniM, StarinieriF, CanepariC, et al.Liver-directed gene therapy with lentiviral vectors in newborn mice and dog puppies. Mol Ther, 2019; 27:323.
90.
VonadaA, NygaardS, GrompeM. Selective expansion of gene-targeted hepatocytes using acetaminophen leads to reproducible long-term liver repopulation. Mol Ther, 2019; 27:326.
91.
MerlinS, FollenziA. Transcriptional targeting and microRNA regulation of lentiviral vectors. Mol Ther Methods Clin Dev, 2019; 12:223–232.
92.
ArgyrosO, WongSP, HarbottleRP. Non-viral episomal modification of cells using S/MAR elements. Expert Opin Biol Ther, 2011; 11:1177–1191.
93.
JacksonDA, JuranekS, LippsHJ. Designing nonviral vectors for efficient gene transfer and long-term gene expression. Mol Ther, 2006; 14:613–626.
94.
FinnJD, SmithAR, PatelMC, et al.A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep, 2018; 22:2227–2235.
