Classical Hereditary galactosemia: findings in patients and animal models.

Classic galactosemia is a rare inborn error of metabolism that affects the metabolism of galactose, a sugar derived from milk and derivates. Classic galactosemia is caused by variants of the GALT gene, which lead to absent or misfolded forms of the ubiquitously present galactose-1-phosphate uridylyltransferase enzyme (GALT) driving galactose metabolites to accumulate, damaging cells from neurons to hepatocytes. The disease has different prevalence around the world due to different allele frequencies among populations and its symptoms range from cognitive and psychomotor impairment to hepatic, ophthalmological, and bone structural damage. The practice of newborn screening still varies among countries, dairy restriction treatment is a consensus despite advances in preclinical treatment strategies. Recent clinical studies in Duarte variant suggest dairy restriction could be reconsidered in these cases. Despite noteworthy advances in the classic galactosemia understanding, preclinical trials are still crucial to fully understand the pathophysiology of the disease and help propose new treatments. This review aims to report a comprehensive analysis of past studies and state of art research on galactosemia screening, its clinical and preclinical trials, and treatments with the goal of shedding light on this complex and multisystemic innate error of the metabolism.

Classic Galactosemia: Clinical and Computational Characterization of a Novel GALT Missense Variant (p.A303D) and a Literature Review.

Classic galactosemia is an autosomal recessive inherited liver disorder of carbohydrate metabolism caused by deficient activity of galactose-1-phosphate uridylyltransferase (GALT). While a galactose-restricted diet is lifesaving, most patients still develop long-term complications. In this study, we report on a two-week-old female patient who is a compound heterozygote for a known pathogenic variant (p.K285N) and a novel missense variant (p.A303D) in the GALT gene. Segregation analysis showed that the patient inherited the p.K285N pathogenic variant from her father and the p.A303D variant from her mother. A bioinformatics analysis to predict the impact of the p.A303D missense variant on the structure and stability of the GALT protein revealed that it may be pathogenic. Based on this finding, we performed a literature review of all GALT missense variants identified in homozygous and compound heterozygous galactosemia patients carrying the p.K285N pathogenic variant to explore their molecular effects on the clinical phenotype of the disease. Our analysis revealed that these missense variants are responsible for a wide range of molecular defects. This study expands the clinical and mutational spectrum in classic galactosemia and reinforces the importance of understanding the molecular consequences of genetic variants to incorporate genetic analysis into clinical care.

Grip strength in patients with galactosemia and in a galactose-1-phosphate uridylyltransferase (GALT)-null rat model.

Classic galactosemia (CG) and clinical variant galactosemia (CVG) are allelic inborn errors of metabolism that result from profound deficiency, and near-profound deficiency, respectively, of galactose-1-P uridylyltransferase (GALT). Despite early detection and lifelong dietary restriction of galactose, which is the current standard of care, most patients with CG/CVG grow to experience a range of long-term developmental and other complications. One of the less well-understood complications of CG/CVG is decreased hand grip strength, as reported by Potter et al. (2013). Here, we confirm this phenotype in an independent cohort of 36 cases (4-18 years) and 19 controls (4-17 years), and further demonstrate that the grip strength deficit observed in cases may be secondary to growth delay. Specifically, we found that when grip strength of cases and controls in a new cohort recruited in 2022 was plotted by weight, rather than age, the difference between cases and controls for both sexes disappeared. Reanalyzing data from the original 2013 cohort, we found that differences in weight accounted for grip strength differences between cases and controls in girls and young women, but not in boys and young men. Finally, we tested whether a GALT-null rat model of CG also showed a grip strength deficit-it did-and again the difference between GALT-null and wild-type rats associated with differences in body mass. Combined, these results confirm that GALT deficiency is associated with a grip strength deficit in both young patients with CG/CVG and GALT-null rats, and further demonstrate that this phenotype may be secondary to growth delay, and therefore not evidence of a muscle abnormality.

Development, optimization and validation of LC-MS/MS method for the determination of DBS GALT enzyme activity.

Galactosemia is a carbohydrate metabolism disorder often caused by galactose-1-phosphate uridyl transferase (GALT) deficiency. Detecting GALT deficiency involves measuring intra-erythrocyte enzyme activity. We aimed to create a robust liquid chromatography-mass spectrometry (LC-MS/MS) method to assess GALT activity in dried blood spot (DBS) samples. We validated this method and compared it to the fluorometric approach. We investigated the impact of K2EDTA and lithium heparin tubes on enzyme activity to identify the best sample collection tube. We also assessed the reaction-stopping method. The developed approach employed [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine as an internal standard (IS). The mean ± SD value for GALT activity of DBS samples was determined as 6.37 ± 1.96 µmol/gHb/hour. The linear range was 0.4-50 µM (2.4-310% of normal) in the DBS method. The % coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. Over 90% recovery was achieved in recovery studies, and no ion suppression from matrix was detected. DBS samples were quite stable for 31 days under different storage conditions. Enzyme activity results reported as <3.5 U/g Hb by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method.

Racial and ethnic diversity of classic and clinical variant galactosemia in the United States.

Classic and clinical variant galactosemia (CG/CVG) are allelic, autosomal recessive disorders that result from deficiency of galactose-1-P uridylyltransferase (GALT). CG/CVG has been reported globally among patients of diverse ancestries, but most large studies of outcomes have included, almost exclusively, patients categorized as White or Caucasian. As a first step to explore whether the cohorts studied are representative of the CG/CVG population at large, we sought to define the racial and ethnic makeup of CG/CVG newborns in a diverse population with essentially universal newborn screening (NBS) for galactosemia: the United States (US). First, we estimated the predicted racial and ethnic distribution of CG/CVG by combining the reported demographics of US newborns from 2016 to 2018 with predicted homozygosity or compound heterozygosity of pathogenic, or likely pathogenic, GALT alleles from the relevant ancestral groups. Incorporating some simplifying assumptions, we predicted that of US newborns diagnosed with CG/CVG, 65% should be White (non-Hispanic), 23% should be Black (non-Hispanic), 10% should be Hispanic, and 2% should be Asian (non-Hispanic). Next, we calculated the observed racial and ethnic distribution of US newborns diagnosed with CG/CVG using available de-identified data from state NBS programs from 2016 to 2018. Of the 235 newborns in this cohort, 41 were categorized as other or unknown. Of the remaining 194, 66% were White (non-Hispanic or ethnicity unknown), 16% were Black (non-Hispanic or ethnicity unknown),15% were Hispanic, and 2% were Asian (non-Hispanic or ethnicity unknown). This observed distribution was statistically indistinguishable from the predicted distribution. To the limits of our study, these data confirm the racial and ethnic diversity of newborns with CG/CVG in the US, demonstrate an approach for estimating CG/CVG racial and ethnic diversity in other populations, and raise the troubling possibility that current understanding of long-term outcomes in CG/CVG may be skewed by ascertainment bias of the cohorts studied.

An Approach for Evaluating Potential Screening Thresholds Using Biomarker Population Distribution and Analytical Imprecision.

BACKGROUND: A common approach in laboratory medicine is to use a simple but sensitive test to screen samples to identify those that require additional investigation with a more complex and informative method. Selection of screening thresholds can be guided by biomarker distribution in the tested population and the analytical imprecision of the method. METHODS: A simulation using joint probabilities derived from the population distribution for galactose-1-phosphate uridylyltransferase (GALT) activity and the analytical imprecision for the GALT assay was used to estimate the number of samples that would require repeat analysis and the number of samples with possibly false-negative screening determinations due to analytical imprecision. RESULTS: In the case of GALT activity, screening a conservative initial threshold 6 standard deviations from the confirmation threshold can essentially eliminate the chance of a false-negative screening determination due to analytical imprecision. The trade-off is a greater number of samples requiring follow-up testing (n = 222, equivalent to 0.15% of samples annually). CONCLUSIONS: Selection of thresholds in a screening algorithm is informed by estimates of the number of samples that would require repeat testing and the number that could be false negative due to analytical imprecision.

Demonstrating the utility of sugar-phosphate phosphatases in coupled enzyme assays: galactose-1-phosphate uridylyltransferase as proof-of-concept.

During our biochemical characterization of select bacterial phosphatases belonging to the haloacid dehalogenase superfamily of hydrolases, we discovered a strong bias of Salmonella YidA for glucose-1-phosphate (Glc-1-P) over galactose-1-phosphate (Gal-1-P). We sought to exploit this ability of YidA to discriminate these two sugar-phosphate epimers in a simple coupled assay that could be a substitute for current cumbersome alternatives. To this end, we focused on Gal-1-P uridylyltransferase (GalT) that is defective in individuals with classical galactosemia, an inborn disorder. GalT catalyzes the conversion of Gal-1-P and UDP-glucose to Glc-1-P and UDP-galactose. When recombinant YidA was coupled to GalT, the final orthophosphate product (generated from selective hydrolysis of Glc-1-P by YidA) could be easily measured using the inexpensive malachite green reagent. When this new YidA-based colorimetric assay was benchmarked using a recombinant Duarte GalT variant, it yielded kcat/Km values that are ~2.5-fold higher than the standard coupled assay that employs phosphoglucomutase and glucose-6-phosphate dehydrogenase. Although the simpler design of our new GalT coupled assay might find appeal in diagnostics, a testable expectation, we spotlight the GalT example to showcase the untapped potential of sugar-phosphate phosphatases with distinctive substrate-recognition properties for measuring the activity of various metabolic enzymes (e.g. trehalose-6-phosphate synthase, N-acetyl-glucosamine-6-phosphate deacetylase, phosphofructokinase).

Addition of galactose-1-phosphate measurement enhances newborn screening for classical galactosemia.

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

Early postnatal alterations in follicular stress response and survival in a mouse model of Classic Galactosemia.

Primary ovarian insufficiency is characterized by accelerated loss of primordial follicles, which results in ovarian failure and concomitant menopause before age 40. About 1-3% of females in the general population are diagnosed with POI; however, greater than 80% of females with the inherited disease Classic Galactosemia will develop POI. Classic Galactosemia is caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase. While dietary restriction of galactose is lifesaving in the neonatal period, the development of complications including primary ovarian insufficiency is not mitigated. Additionally, the pattern(s) of follicle loss have not been completely characterized. The chronic accumulation of aberrant metabolites such as galactose-1-phosphate and galactitol are suspected culprits in the development of the sequelae, yet the mechanisms remain elusive.Our group uses a GalT gene-trapped mouse model to study the pathophysiology of primary ovarian insufficiency in Classic Galactosemia. We recently showed that differences in the Integrated Stress Response pathway occur in mutant ovaries that likely contribute to their primary ovarian insufficiency phenotype. Using immunofluorescent staining of histological sections of ovaries at progressive ages, we saw evidence of altered Integrated Stress Response activity in granulosa cells and primordial oocytes consistent with accelerated primordial follicle growth activation, aberrant DNA damage and/or repair, and increased cellular stress/death. Overall, our findings indicate that abnormal Integrated Stress Response in the Classic Galactosemia model ovary results in accelerated primordial follicle growth activation, sometimes referred to as "burnout." These aberrant early events help further clarify when/how the primary ovarian insufficiency phenotype arises under galactosemic conditions.

A multinational study of acute and long-term outcomes of Type 1 galactosemia patients who carry the S135L (c.404C > T) variant of GALT.

Patients with galactosemia who carry the S135L (c.404C > T) variant of galactose-1-P uridylyltransferase (GALT), documented to encode low-level residual GALT activity, have been under-represented in most prior studies of outcomes in Type 1 galactosemia. What is known about the acute and long-term outcomes of these patients, therefore, is based on very limited data. Here, we present a study comparing acute and long-term outcomes of 12 patients homozygous for S135L, 25 patients compound heterozygous for S135L, and 105 patients homozygous for two GALT-null (G) alleles. This is the largest cohort of S135L patients characterized to date. Acute disease following milk exposure in the newborn period was common among patients in all 3 comparison groups in our study, as were long-term complications in the domains of speech, cognition, and motor outcomes. In contrast, while at least 80% of both GALT-null and S135L compound heterozygous girls and women showed evidence of an adverse ovarian outcome, prevalence was only 25% among S135L homozygotes. Further, all young women in this study with even one copy of S135L achieved spontaneous menarche; this is true for only about 33% of women with classic galactosemia. Overall, we observed that while most long-term outcomes trended milder among groups of patients with even one copy of S135L, many individual patients, either homozygous or compound heterozygous for S135L, nonetheless experienced long-term outcomes that were not mild. This was true despite detection by newborn screening and both early and life-long dietary restriction of galactose. This information should empower more evidence-based counseling for galactosemia patients with S135L.

Multi-omics in classical galactosemia: Evidence for the involvement of multiple metabolic pathways.

Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.

Harnessing the Power of Purple Sweet Potato Color and Myo-Inositol to Treat Classic Galactosemia.

Classic Galactosemia (CG) is a devastating inborn error of the metabolism caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase in galactose metabolism. Severe complications of CG include neurological impairments, growth restriction, cognitive delays, and, for most females, primary ovarian insufficiency. The absence of the GALT enzyme leads to an accumulation of aberrant galactose metabolites, which are assumed to be responsible for the sequelae. There is no treatment besides the restriction of dietary galactose, which does not halt the development of the complications; thus, additional treatments are sorely needed. Supplements have been used in other inborn errors of metabolism but are not part of the therapeutic regimen for CG. The goal of this study was to test two generally recognized as safe supplements (purple sweet potato color (PSPC) and myo-inositol (MI)) that may impact cellular pathways contributing to the complications in CG. Our group uses a GalT gene-trapped mouse model to study the pathophysiology in CG, which phenocopy many of the complications. Here we report the ability of PSPC to ameliorate dysregulation in the ovary, brain, and liver of our mutant mice as well as positive results of MI supplementation in the ovary and brain.

Pathophysiology of long-term complications in classic galactosemia: What we do and do not know.

Despite many decades of research involving both human subjects and model systems, the underlying pathophysiology of long-term complications in classic galactosemia (CG) remains poorly understood. In this review, intended for those already familiar with galactosemia, we focus on the big questions relating to outcomes, mechanism, and markers, drawing on relevant literature where available, attempting to navigate inconsistencies where they appear, and acknowledging gaps in knowledge where they persist.

Galactosemia: Biochemistry, Molecular Genetics, Newborn Screening, and Treatment.

Galactosemia is an inborn disorder of carbohydrate metabolism characterized by the inability to metabolize galactose, a sugar contained in milk (the main source of nourishment for infants), and convert it into glucose, the sugar used by the body as the primary source of energy. Galactosemia is an autosomal recessive genetic disease that can be diagnosed at birth, even in the absence of symptoms, with newborn screening by assessing the level of galactose and the GALT enzyme activity, as GALT defect constitutes the most frequent cause of galactosemia. Currently, galactosemia cannot be cured, but only treated by means of a diet with a reduced content of galactose and lactose. Although the diet is able to reverse the neonatal clinical picture, it does not prevent the development of long-term complications. This review provides an overview of galactose metabolism, molecular genetics, newborn screening and therapy of galactosemia. Novel treatments for galactosemia currently being investigated in (pre)clinical studies and potentially able to prevent long-term complications are also presented.

Novel mRNA therapy restores GALT protein and enzyme activity in a zebrafish model of classic galactosemia.

Messenger RNA (mRNA) has emerged as a novel therapeutic approach for inborn errors of metabolism. Classic galactosemia (CG) is an inborn error of galactose metabolism caused by a severe deficiency of galactose-1-phosphate:uridylyltransferase (GALT) activity leading to neonatal illness and chronic impairments affecting the brain and female gonads. In this proof of concept study, we used our zebrafish model for CG to evaluate the potential of human GALT mRNA (hGALT mRNA) packaged in two different lipid nanoparticles to restore GALT expression and activity at early stages of development. Both one cell-stage and intravenous single-dose injections resulted in hGALT protein expression and enzyme activity in the CG zebrafish (galt knockout) at 5 days post fertilization (dpf). Moreover, the levels of galactose-1-phosphate (Gal-1-P) and galactonate, metabolites that accumulate because of the deficiency, showed a decreasing trend. LNP-packaged mRNA was effectively translated and processed in the CG zebrafish without signs of toxicity. This study shows that mRNA therapy restores GALT protein and enzyme activity in the CG zebrafish model, and that the zebrafish is a suitable system to test this approach. Further studies are warranted to assess whether repeated injections safely mitigate the chronic impairments of this disease.

Qualitative interviews with adults with Classic Galactosemia and their caregivers: disease burden and challenges with daily living.

BACKGROUND: Classic Galactosemia is a rare, autosomal recessive disease in which galactose is not metabolized properly due to severe deficiency/absence of the galactose-1-phosphate uridylyltransferase (GALT) enzyme, converting to an aberrant and toxic metabolite, galactitol. Newborn screening and timely galactose-restricted diet can resolve acute symptoms and decrease fatalities. However, despite this, significant chronic, progressive morbidities remain which have a real impact upon daily life. To better understand the burden of disease, 20 in-depth qualitative interviews were undertaken with adult patients (n = 12), and their caregivers (n = 8), enrolled in the ACTION-Galactosemia trial, part of a clinical program designed to investigate the safety and efficacy of AT-007 (govorestat) in reducing toxic galactitol and long-term clinical outcomes in Classic Galactosemia. RESULTS: Interviews revealed the substantial burden of Classic Galactosemia on patients and families. Most adults were not able to live independently, and all required support with day-to-day activities. Short- and long-term memory difficulties and tremors were identified as the most frequently experienced and challenging symptoms. Other difficulties such as fine motor skills and slow/slurred speech contribute to the significant impact on daily activities, affecting ability to communicate and interact with others. Symptoms were first noticed in early childhood and worsened with age. Classic Galactosemia impacted all areas of daily functioning and quality of life, leading to social isolation, anxiety, anger/frustration and depression. This demonstrates the significant burden of disease and challenges associated with Classic Galactosemia. CONCLUSIONS: The impact on both patients and caregivers underscores the severity of the unmet medical need and the importance of pharmacological intervention to halt or prevent disease progression. Any treatment that could reduce symptoms or slow functional decline would ease the burden of this condition on patients and caregivers.

Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin - an inhibitor of the de novo sphingolipid synthesis pathway - inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.

The Importance of Neonatal Screening for Galactosemia.

Galactosemia is an inborn metabolic disorder caused by a deficient activity in one of the enzymes involved in the metabolism of galactose. The first description of galactosemia in newborns dates from 1908, ever since complex research has been performed on cell and animal models to gain more insights into the molecular and clinical bases of this challenging disease. In galactosemia, the newborn appears to be born in proper health, having a window of opportunity before developing major morbidities that may even be fatal following ingestion of milk that contains galactose. Galactosemia cannot be cured, but its negative consequences on health can be avoided by establishing precocious diagnosis and treatment. All the foods that contain galactose should be eliminated from the diet when there is a suspicion of galactosemia. The neonatal screening for galactosemia can urge early diagnosis and intervention, preventing complications. All galactosemia types may be detected during the screening of newborns for this disorder. The major target is, however, galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia, which is diagnosed by applying a combination of total galactose and GALT enzyme analysis as well as, in certain programs, mutation screening. Most critically, infants who exhibit symptoms suggestive of galactosemia should undergo in-depth testing for this condition even when the newborn screening shows normal results. The decision to enroll global screening for galactosemia among the specific population still faces many challenges. In this context, the present narrative review provides an updated overview of the incidence, clinical manifestations, diagnosis, therapy, and prognosis of galactosemia, questioning under the dome of these aspects related to the disease the value of its neonatal monitoring.

Neonatal GALT gene replacement offers metabolic and phenotypic correction through early adulthood in a rat model of classic galactosemia.

Classic galactosemia (CG) results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection by newborn screening and lifelong dietary restriction of galactose, most patients grow to experience a range of long-term complications. Recently, we developed and characterized a GALT-null rat model of CG and demonstrated that AAV9-hGALT, administered by tail vein injection to neonatal pups, dramatically improved plasma, liver, and brain galactose metabolites at 2 weeks posttreatment. Here we report a time-course study of GALT restoration in rats treated as neonates with scAAV9-hGALT and harvested at 8, 14, 30, and 60 days. Cohorts of rats in the two older groups were weaned to diets containing either 1% or 3% of calories from galactose. As expected, GALT activity in all treated animals peaked early and then diminished over time, most notably in liver, ostensibly due to dilution of the nonreplicating episomal vector as transduced cells divided. All treated rats showed dramatic metabolic rescue through 1 month, and those weaned to the lower galactose diet showed continued strong metabolic rescue into adulthood (2 months). Prepubertal growth delay and cataracts were both partially rescued by treatment. Finally, we found that UDP glucose pyrophosphorylase (UGP), which offers a metabolic bypass around missing GALT, was 3-fold more active in brain samples from adult rats than from young pups, offering a possible explanation for the improved ability of older GALT-null rats to metabolize galactose. Combined, these results document promising metabolic and phenotypic efficacy of neonatal GALT gene replacement in a rat model of classic galactosemia.

AAV-mediated expression of galactose-1-phosphate uridyltransferase corrects defects of galactose metabolism in classic galactosemia patient fibroblasts.

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose-1-phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno-associated virus (AAV)-mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV-mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFßRII protein levels and that these effectors of cellular homeostasis could be restored following AAV-mediated expression of GALT. Finally, we show initial in vivo proof-of-concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV-GALT.