Fig 1: Behavioral testing in Gamt deficiency reveals a learning deficit, resolved with hepatic GAMT gene therapyBehavioral testing was performed at 8 months of age (n = 8 per group). Untreated Gamt−/− mice showed deficits in learning, with an increase in primary latency to the escape hole (A and B), resolving with AAV-based gene therapy. Differences were present in distance traveled during acquisition (C and D). Short-term reference memory during the probe trial at 24 h showed no statistically significant difference in latency or distance traveled between the groups (E and F). However, at 1 week, testing longer term memory, primary latency for the untreated Gamt−/− mice was prolonged (G), but there was no difference in distance traveled (H). Using 1 of 3 search strategies in the Barnes maze (I), Gamt+/+ and treated Gamt−/− mice switch from using primarily non-hippocampal random (gray) and serial (orange) strategies to a more hippocampal-based direct strategy (blue) (J–M). In contrast, untreated GAMT−/− mice rely primarily on the serial search strategy, with reduced spatial pathway activity. In the probe trial at 24 h (O), untreated Gamt-deficient mice have reduced direct pathway activity, and by 1 week (P), a test of longer-term memory, the direct strategy is absent, unlike WT or treated Gamt−/− mice. Error bars represent mean +/- standard deviation.
Fig 2: AAV expressing human codon-optimized GAMT results in improvement in tissue levels of GAA while increasing creatine concentrations in male and female Gamt-deficient miceGAA levels are markedly elevated in the brain (A), heart (B), kidney (C), liver (D), and skeletal muscle (E) of untreated Gamt−/− mice (red data points). Normalization of elevated GAA occurs with AAV-based hepatic gene therapy in the heart, liver, and skeletal muscle, with marked reductions in the brain and kidney. Creatine is nearly undetectable in brain (F), heart (G), kidney (H), liver (I), and skeletal muscle (J) in untreated Gamt−/− mice (red data points). Tissue levels are markedly improved, with complete restoration in the brain, heart and skeletal muscle in treated mice. Values represent means ± standard deviations. n = 3–8 per group. Red represents untreated Gamt−/− mice; blue represents treated Gamt−/− mice; black represents WT controls.
Fig 3: Increasing AAV dose results in augmented GAMT expression and biochemical responseAdult Gamt−/− mice were administered AAVrh10 expressing human codon-optimized GAMT intravenously and analyzed 30 days after the injection. RNA expression (A–E) by in situ hybridization (RNAscope) is demonstrated by increasing red precipitate in these representative images (A, untreated; B, 5 × 1012 GC/kg; C, 1 × 1013 GC/kg; D, 5 × 1013 GC/kg; E, 1 × 1014 GC/kg). Protein expression simultaneously increases as detected by immunohistochemical detection and DAB staining (F–J) in these representative images (F, untreated; G, 5 × 1012 GC/kg; H, 1 × 1013 GC/kg; I, 5 × 1013 GC/kg; J, 1 × 1014 GC/kg). With increasing doses, the plasma biochemical response to hepatic hcoGAMT expression increases; plasma creatine incrementally increases and normalizes (K) as elevated plasma guanidinoacetic acid levels abate (L). Values represent means ± standard deviations. (F) and (L) n = 5 per group. Red represents baseline levels in Gamt−/− mice. Blue represents plasma values at 30 days after AAV administration. Black dotted line demonstrates mean plasma creatine level (with range of minimum and maximum represented with gray dotted line) in (K) and mean plasma guanidinoacetic acid level in (L). GAA, guanidinoacetic acid. Bar size in (F) and (G), 100 μm.
Fig 4: Dose-finding studies demonstrate quantitative increases in GAMT expressionAdult Gamt−/− mice were intravenously administered AAVrh10 expressing human codon-optimized GAMT and analyzed 30 days after the injection. AAV DNA copy-number analysis (A) per diploid nucleus of the liver, RNA expression (B) by quantitative real-time PCR of human codon-optimized GAMT, and GAMT protein expression (C and D) demonstrate increases with escalating dose. (RT-PCR, n = 5 per group; quantitative western blot analysis, n = 3 per group). (C shows representative images). Values represent means ± standard deviations. GC, genome copies; GAMT, guanidino N-methyltransferase.
Fig 5: AAV administration results in long-term hepatic genome persistence and GAMT protein expressionMice were euthanized 1 year after intravenous administration of AAVrh10 expressing hcoGAMT under a liver-specific promoter. Hepatic AAV copy-number analysis (A) demonstrates AAV genome persistence. Representative images of immunohistochemistry specific for human GAMT protein show, for comparison, widespread expression in human liver (B), no GAMT expression in WT murine hepatocytes (C), and hepatic GAMT expression, most prominently in hepatocytes near the hepatic veins, in both female (D) and male (E) AAV-treated Gamt−/− mice. Western blotting (F) demonstrates the specificity of antibody for human GAMT and evidence of hepatic expression in AAV-treated GAMT−/− mice. Plasma alanine aminotransferase (G) and aspartate aminotransferase (H) were determined in WT-treated Gamt−/−, and untreated Gamt−/− mice. Values represent means ± standard deviations. For (A), n = 4–8 per group; for (G) and (H), n = 4 per group. AAV, adeno-associated virus.
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