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P. Bianchi and E. Fermo
between the type of mutations and the outcome of the treatment.
Allosteric activator (AG-348)
AG-348 is an allosteric activator of PK-R that binds in a pocket at the dimer-dimer interface, distinct from the allosteric activator fructose 1,6 bisphosphate binding domain, inducing the active R-state conformation of the PK-R tetramer.
Preclinical studies showed that AG-348 enhanced activity in vitro in wild-type PK and in a broad spectrum of PKLR mutations; this finding was consistent with the known binding site for AG-348, which is distinct from the areas of the most common PKLR mutations.11 Data from phase I and phase II studies demonstrated that the glycolytic path- way is activated upon treatment with AG-348, and that 54% of PK-deficient subjects experienced a rise in hemoglo- bin, all of whom had at least one missense mutation.54,96 It has therefore been hypothesized that a minimal level of full-length PK protein is required for enzyme activation, excluding patients carrying two nonsense variants from the potential benefits of the treatment.54 In an AG-348 clinical trial, evaluating p.R479H as a splicing variant other than a simple amino acid substitution caused an increase of the percentage of patients with a hemoglobin response from 48% to 54%.54
A more recent study investigated the effect of ex vivo treatment with AG-348 on enzyme activity, thermostabili- ty, protein levels and ATP in PK-deficient red cells from 15 patients with different genotypes, including the most fre- quently reported variants in Caucasian p.R486W and p.R510Q;97 the overall results showed a mean 1.8-fold increase in PK activity and a 1.5-fold increase in ATP levels. Protein analyses suggested that a sufficient level of protein is required for cells to respond to AG-348 treatment, as pre- viously reported.54 Interestingly, the thermostability of PK was also found to be significantly improved upon ex vivo treatment with AG-348, but with a high variability in response among the different genotypes; this was particu- larly evident in PK patients carrying the common mutation p.R510Q, which is known to affect catalytic activity only slightly, but to be highly unstable.13 Overall, these data demonstrated that the clinical utility of AG-348 in PK-defi- cient patients is influenced by the type of mutations, and that variability in the response can also be increased by the compound heterozygosity that is present in most patients. Prospective studies in patients across a broader range of genotypes and disease severity are required to identify patients who can benefit most from the treatment.
Gene therapy
Ex vivo gene therapy for hematologic genetic disorders is becoming a reality in clinical practice. This gene thera- py strategy is based on an autologous transplant in which the infused cells are genetically corrected ex vivo. There are several ongoing clinical trials on the use of gene therapy for rare anemias (β-thalassemia, sickle cell disease, Fanconi anemia) and hereditary metabolic dis- eases.98,99 Preclinical studies focused on the treatment of PK deficiency by gene therapy have been successfully performed.100-102 Autologous cells corrected with a lentivi- ral vector carrying a codon optimized version of the wild-type cDNA sequence of the PK-R gene have been demonstrated to be able to compensate the disease phe- notype in a murine model, without any adverse effect related to the procedure.102 This procedure has been des- ignated as an orphan drug by the European Medicines Agency (EU/3/14/1330; https://goo.gl/T4N6mO) and by the U.S. Food and Drug Administration (DRU-2016- 5168).103
An open-label, phase 1 gene therapy study consisting of autologous hematopoietic stem and progenitor cells transduced ex vivo with a lentiviral vector encoding for the PK enzyme has been approved and recently opened. PK-deficient patients with confirmed genotype and severe, transfusion-dependent anemia despite splenecto- my may be eligible for enrollment (www.clinicaltrial.gov NCT04105166).
Conclusions
One of the clear advantages of NGS technologies is the availability of molecular testing for rare diseases in many laboratories, resulting in increased awareness of rare con- genital conditions, in the dramatically increased number of molecular variants, in the reduced time of diagnosis and number of misdiagnoses. However, the huge amount of data obtained should be interpreted in the light of knowledge of the pathogenic basis of diseases and always supported by functional studies: on top of the molecular lesion itself, the effect of mutations on the expression and functionality of the protein is known for only a few variants. In addition, the study of compensa- tory effects of other metabolic pathways and cellular involvement (e.g., membrane channel activities, mem- brane stability) in response to energy depletion will offer new insights into the interpretation of the effect of PKLR mutations and phenotype.
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