ST3GAL5 Human

What is ST3GAL5 and what is its primary function in human biology?

ST3GAL5 (also known as GM3 synthase, GM3S, or SIAT9) is the gene encoding the rate-limiting enzyme for production of all a- and b-series gangliosides normally enriched in mammalian brain. It catalyzes the transfer of sialic acid to lactosylceramide, forming GM3, which serves as the precursor for complex ganglioside synthesis. GM3 and derivative gangliosides are expressed in cytosolic membranes of all mammalian cells, where they contribute to microdomain architecture and activity of intramembrane proteins. The gene produces multiple protein isoforms that differ in their N-termini due to alternative translation initiation sites.

What are the main isoforms of ST3GAL5 and how do they differ functionally?

ST3GAL5 exists in multiple isoforms that differ in their N-termini. The ST3GAL5-1a-2 (NM_003896) is the most abundant mRNA variant in human brain among four mRNA variants. This variant has a first AUG start codon in a weak translation initiation context (AUUAGUAUGC), causing most ribosomes to skip it and recognize either of two downstream AUG sequences as the start codon. This results in three ST3GAL5 protein isoforms with different N-termini. Research has shown that M3-ST3GAL5 (the shortest isoform) is the most stable, though all isoforms can function in ganglioside synthesis when expressed in cell culture models. Adding a Kozak sequence (GCCACC) greatly enhances expression of the shortest construct.

How does ST3GAL5 deficiency manifest clinically in humans?

Biallelic loss-of-function mutations in ST3GAL5 result in GM3 synthase deficiency (GM3SD), characterized by infantile-onset epileptic encephalopathy, auditory and visual impairment, global psychomotor delay, extrapyramidal movements, and untimely death. The condition manifests with complete absence of GM3 and its downstream derivatives in plasma and brain tissue, leading to systemic ganglioside deficiency. In North American Old Order Amish communities, GM3SD occurs at an incidence of approximately 1 per 1,200 births due to a founder variant (ST3GAL5 c.862C > T; p.Arg288Ter) that abrogates ST3GAL5 activity.

What experimental models are currently available for studying ST3GAL5 function and deficiency?

Several complementary models are used to study ST3GAL5 function:

• Patient-derived iPSCs differentiated into cortical neurons: These provide a human cellular model that accurately reflects the genetic background of patients with ST3GAL5 deficiency. This model allows for direct testing of gene replacement constructs for their ability to reconstitute ganglioside synthesis.

• St3gal5-/- knockout mice: These mice show tissue deficiency of GM3 but present with a milder phenotype than human patients, primarily exhibiting hearing loss.

• St3gal5-/-/B4galnt1-/- double knockout mice: These exhibit more severe neuropathology and functional deficits concordant with human GM3SD, providing a more phenotypically relevant murine model.

• HeLa cell culture: Used for initial validation of ST3GAL5.v1 and ST3GAL5.v2 constructs and testing their expression.

Each model has limitations - particularly the single knockout mice which underrepresent the severity of human disease.

What techniques are employed to measure ST3GAL5 activity and ganglioside production?

The measurement of ST3GAL5 activity and ganglioside production typically involves multiple techniques:

• Protein expression analysis: Western blotting to detect ST3GAL5 protein expression in tissues and cell cultures.

• Ganglioside profiling: Thin-layer chromatography or high-performance liquid chromatography coupled with mass spectrometry to detect and quantify GM3 and downstream gangliosides like GD1a, GD1b, and GT1b.

• Gene expression analysis: RT-PCR or RNA sequencing to measure mRNA levels of ST3GAL5 and related genes.

• Promoter activity assays: Luciferase reporter assays to evaluate the impact of promoter variants on gene expression.

• Viral vector-mediated gene delivery: Used both in vitro and in vivo to assess the functional rescue of ST3GAL5 deficiency.

How do researchers distinguish between changes in ganglioside profiles due to ST3GAL5 deficiency versus other pathways?

Researchers use several approaches to attribute changes in ganglioside profiles specifically to ST3GAL5 deficiency:

• Comparative analysis: Comparing ganglioside profiles between wild-type, ST3GAL5-deficient, and ST3GAL5-restored samples allows researchers to identify ST3GAL5-dependent gangliosides.

• Alternative pathway analysis: In the absence of GM3 synthase, lactosylceramide (LacCer) is shunted into alternative biosynthetic pathways for producing O-series gangliosides. Measuring these alternative gangliosides helps understand compensatory mechanisms.

• Double knockout models: Using St3gal5-/-/B4galnt1-/- double knockout mice helps distinguish between effects due to ST3GAL5 deficiency alone versus combined pathway disruptions.

• Rescue experiments: Restoring ST3GAL5 expression and observing the normalization of specific gangliosides confirms their dependence on the enzyme.

What are the most significant ST3GAL5 genetic variants identified in human populations?

Several pathogenic variants have been linked to GM3 synthase deficiency syndrome worldwide. In Old Order Amish communities of North America, there is an enriched incidence of GM3SD (approximately 1 per 1,200 births) due to a severe ST3GAL5 c.862C > T (p.Arg288Ter) founder variant that abrogates ST3GAL5 activity and results in absence of GM3 and its most important downstream products (GM1, GD1a, GD1b, and GT1b).

How is ST3GAL5 expression regulated at the transcriptional level?

ST3GAL5 expression is regulated by several transcription factors. Research has identified that activating protein-1 (AP-1), NKX3.1, and specificity protein 1 (SP1) play roles in the transcriptional regulation of ST3GAL5. Promoter activity was significantly decreased in three promoter haplotypes of ST3GAL5 and increased in one promoter haplotype, as determined by functional characterization studies.

The transcriptional regulation of ST3GAL5 is complex and tissue-specific, with different promoter haplotypes showing varying activity levels. This regulation can affect the production of gangliosides and potentially contribute to disease pathogenesis when altered.

What methodologies should be employed to characterize novel ST3GAL5 variants?

To characterize novel ST3GAL5 variants, researchers should employ a multi-faceted approach:

• Genetic screening: Sequencing the ST3GAL5 gene in patient and control populations to identify variants.

• Bioinformatic analysis: Using prediction tools to assess the potential impact of variants on protein function.

• Functional characterization:

  • Promoter activity assays using luciferase reporters

  • Electrophoretic mobility shift assays to identify transcription factor binding

  • Expression analysis in relevant cell types

• Haplotype analysis: Assembling variants into haplotypes and assessing their functional significance.

• Population studies: Determining the frequency of variants in different populations.

• Phenotype correlation: Analyzing correlations between functional haplotypes and clinical characteristics of patients.

• In vitro enzymatic assays: Testing the activity of variant ST3GAL5 proteins using appropriate substrates.

How does ST3GAL5 deficiency affect ganglioside biosynthesis pathways in humans versus animal models?

ST3GAL5 deficiency affects ganglioside biosynthesis differently in humans compared to mouse models:

In humans with severe, biallelic loss-of-function mutations in ST3GAL5:

• Complete absence of GM3 and its downstream derivatives in plasma and brain tissue

• Epileptic encephalopathy and psychomotor stagnation within months of life

• Severe neurological impairment including auditory and visual deficits

In St3gal5-/- mice:

• Tissue deficiency of GM3 but a comparatively mild phenotype

• Primary documented phenotype is hearing loss

• Lactosylceramide is shunted into alternative biosynthetic pathways for O-series gangliosides

This discrepancy may be due to species differences in ganglioside biology or compensatory mechanisms. Complex gangliosides may have redundant functions in maintaining membrane physics and signal transduction that vary between species. The St3gal5-/-/B4galnt1-/- double knockout mouse provides a more severe phenotype that better matches human disease, suggesting multiple pathways may compensate for single enzyme deficiencies in mice.

What are the cellular consequences of altered ganglioside profiles due to ST3GAL5 dysfunction?

Altered ganglioside profiles due to ST3GAL5 dysfunction result in widespread cellular consequences:

• Membrane structure alterations: Gangliosides contribute to microdomain architecture in cytosolic membranes, and their absence disrupts membrane organization.

• Signaling pathway disruption: Gangliosides interact with and modulate intramembrane proteins involved in signaling, affecting numerous cellular processes.

• Neurological development impairment: GM3 and derivative gangliosides are enriched in the brain and are crucial for normal neurological development and function.

• Metabolic rerouting: In the absence of ST3GAL5 activity, precursors like lactosylceramide are redirected to alternative pathways, potentially causing accumulation of other glycosphingolipids.

• Altered cell-cell interactions: Gangliosides participate in cell recognition and adhesion processes, affecting cellular communication.

These disruptions collectively contribute to the severe neurological phenotype observed in patients with GM3 synthase deficiency.

Why do humans and mice with ST3GAL5 deficiency show different disease severity?

The different disease severity between ST3GAL5-deficient humans and mice represents an important experimental challenge. Several factors may contribute to this discrepancy:

• Compensatory pathways: Mice may have more robust compensatory pathways that mitigate the effects of ST3GAL5 deficiency. In both human patients and St3gal5-/- mice, lactosylceramide is shunted into alternative biosynthetic pathways for O-series gangliosides, but the effectiveness of this compensation may differ.

• Developmental timing: The timing of ganglioside requirement during neurodevelopment may differ between species.

• Functional redundancy: Complex gangliosides may have redundant functions for maintaining membrane physics and signal transduction that vary between species.

• Metabolic differences: Different metabolic rates and lipid utilization between species may affect the impact of ganglioside deficiency.

Research suggests that to create a more phenotypically relevant murine model requires simultaneous disruption of two serial enzymes in the ganglioside synthetic pathway: St3gal5 and B4galnt1. These double knockout mice exhibit severe neuropathology and functional deficits more concordant with human GM3SD.

What gene therapy strategies show promise for treating ST3GAL5 deficiency?

Recent research has demonstrated several promising gene therapy strategies for treating ST3GAL5 deficiency:

• rAAV-mediated ST3GAL5 gene replacement: Recombinant adeno-associated viruses (rAAVs) can cross the blood-brain barrier to induce widespread, long-term gene expression in the CNS.

• Spatially controlled expression: Optimization of transgene expression using:

  • Neuron-specific promoters (human Synapsin1)

  • Liver-specific miRNA targeting sequences (miR-122) to prevent off-target expression

  • Self-complementary AAV (scAAV) design for faster and higher gene expression

• Optimized delivery routes: Intracerebroventricular (ICV) injection has shown superior efficacy compared to intravenous administration at clinically feasible doses.

These approaches have demonstrated restoration of cerebral ganglioside synthesis, amelioration of neuropathology, and improvement in motor function in mouse models of GM3SD, supporting further clinical development of ST3GAL5 gene therapy.

What are the key considerations for designing effective ST3GAL5 replacement vectors?

Designing effective ST3GAL5 replacement vectors requires careful attention to several key factors:

• Transgene design:

  • Selection of appropriate isoform (M3-ST3GAL5 is the most stable)

  • Codon optimization for enhanced expression

  • Addition of Kozak sequence to improve translation efficiency

  • Consideration of transgene size for compatibility with self-complementary AAV vectors (<2.5 kb)

• Spatial regulation:

  • Cell-specific promoters (e.g., human Synapsin1 for neuronal expression)

  • miRNA targeting sequences (e.g., miR-122 sites to silence expression in hepatocytes)

  • Balancing transcriptional and post-transcriptional regulation

• Vector selection:

  • AAV9 can cross the blood-brain barrier

  • Self-complementary AAV (scAAV) allows for faster and higher gene expression

  • Vector manufacturing considerations (avoiding transgene toxicity in production cells)

• Delivery considerations:

  • Route of administration (ICV vs. intravenous)

  • Dose optimization to achieve therapeutic effect while minimizing toxicity

  • Timing of intervention

These design elements have proven critical for achieving safe and effective gene therapy for ST3GAL5 deficiency in preclinical models.

How does the administration route affect the efficacy of ST3GAL5 gene therapy?

The administration route significantly impacts the efficacy of ST3GAL5 gene therapy, with research comparing intracerebroventricular (ICV) and intravenous (i.v.) routes:

ICV injection advantages:

• Bypasses the blood-brain barrier

• Achieves promising therapeutic outcomes at clinically feasible doses (2 × 10^13 gcs/kg)

• More effectively restores ganglioside production and prevents disease manifestations

• Similar in principle to intrathecal injection used for other CNS-directed therapies

Intravenous injection limitations:

• Requires higher doses (10-fold higher: 2 × 10^14 gcs/kg)

• Less effective at restoring ganglioside production or preventing disease manifestations

• Potential for off-target expression and toxicity (particularly hepatotoxicity with ubiquitous promoters)

What safety concerns must be addressed in developing ST3GAL5 gene therapy?

Development of ST3GAL5 gene therapy must address several important safety concerns:

• Hepatotoxicity: First-generation vectors using ubiquitous promoters caused fatal hepatotoxicity when administered systemically. This was manifested as liver inflammation, hepatocyte death, and severe transcriptomic derangements.

• Off-target expression: Overexpression of ST3GAL5 in non-target tissues can lead to unpredictable consequences and toxicity.

• Manufacturing challenges: High transgene expression during vector production may cause toxicity in HEK293 cells, resulting in low vector titers.

• Dosing considerations: Determining the minimal effective dose while avoiding toxicity is crucial for clinical translation.

• Long-term effects: Ensuring persistent and appropriately regulated transgene expression without causing developmental disruptions.

These concerns have been addressed through:

• CNS-restricted expression using neuron-specific promoters

• Liver-detargeted expression using miR-122 binding sites

• Optimized vector design to enhance manufacturing

This spatially regulated approach eliminated both hepatotoxicity and manufacturing bottlenecks, creating a clinically translatable candidate.

How might single-cell analysis inform our understanding of ST3GAL5 function in different cell types?

Single-cell analysis represents a frontier approach that could significantly advance our understanding of ST3GAL5 function across different cell types:

• Cell-type specific expression patterns: Single-cell RNA sequencing could reveal differential expression of ST3GAL5 across neural and non-neural cell types, identifying cells most dependent on its function.

• Cell-autonomous vs. non-cell-autonomous effects: Determining whether the pathology of ST3GAL5 deficiency results from dysfunction within specific cells or from disrupted intercellular interactions.

• Compensatory mechanism identification: Single-cell analysis could identify cell-specific compensatory pathways activated in response to ST3GAL5 deficiency, explaining differential vulnerability.

• Developmental trajectory mapping: Tracking ST3GAL5 expression and ganglioside profiles throughout development at single-cell resolution could pinpoint critical developmental windows.

• Therapeutic targeting precision: Informing more precise therapeutic targeting by identifying the most critical cell populations requiring ST3GAL5 restoration.

This approach could help resolve the discrepancy between mouse and human phenotypes and guide more effective therapeutic strategies.

What are the challenges in translating ST3GAL5 gene therapy findings from mouse models to humans?

Translating ST3GAL5 gene therapy findings from mouse models to humans faces several significant challenges:

• Species differences in ganglioside biology: Mice and humans show developmental and functional differences in ganglioside biology, with mice exhibiting milder phenotypes from ST3GAL5 deficiency.

• Model limitations: Current mouse models (St3gal5-/- and St3gal5-/-/B4galnt1-/-) either underrepresent the disease severity or have confounding factors from multiple gene disruptions.

• Anatomical differences: Differences in brain size, structure, and development between mice and humans affect vector distribution and therapeutic efficacy.

• Dosing translation: Scaling up vector doses from mice to humans while maintaining safety and efficacy presents significant challenges.

• Timing of intervention: The optimal timing for therapeutic intervention may differ between species due to differences in developmental timelines.

Researchers suggest that modeling GM3SD in larger gyrencephalic species, such as pigs or sheep, might prove more informative for future translational studies, as these animals may better represent human ganglioside biology and brain structure.

How might combinatorial gene therapy approaches improve outcomes for complex ganglioside disorders?

Combinatorial gene therapy approaches could significantly improve outcomes for complex ganglioside disorders through several mechanisms:

• Multiple enzyme restoration: Co-delivery of ST3GAL5 with other enzymes in the ganglioside synthesis pathway, such as B4GALNT1, could more completely restore ganglioside profiles. Research has shown that co-injection of St3gal5-/-/B4galnt1-/- mice with both ST3GAL5 and B4GALNT1 rAAV vectors completely eliminated behavior impairments.

• Pathway optimization: Targeting multiple enzymes could optimize ganglioside synthesis by preventing metabolite accumulation or shunting into alternative pathways.

• Synergistic effects: Simultaneous restoration of multiple enzymes might produce synergistic therapeutic effects beyond what could be achieved with single gene replacement.

• Personalized approaches: Combinatorial approaches could be tailored to specific genetic profiles of patients with complex ganglioside disorders.

• Reduced dose requirements: Lower doses of each individual vector might be effective when used in combination, potentially reducing toxicity concerns.

These combinatorial approaches may be particularly valuable for addressing complex ganglioside disorders involving multiple enzyme deficiencies or for optimizing therapeutic outcomes in conditions with partial deficiencies.

What are the key journals and conferences for following advances in ST3GAL5 research?

Researchers interested in ST3GAL5 should monitor publications in journals focusing on neuroscience, genetics, glycobiology, and gene therapy. Based on the current literature, key resources include:

• Journals:

  • Journal of Clinical Investigation

  • Nature Medicine

  • Science Translational Medicine

  • Human Molecular Genetics

  • Molecular Therapy

  • Journal of Biological Chemistry

  • Glycobiology

  • Journal of Lipid Research

• Conferences:

  • American Society of Gene & Cell Therapy (ASGCT) Annual Meeting

  • Society for Glycobiology Annual Meeting

  • International Society for Neurochemistry Conference

  • American Society of Human Genetics Annual Meeting

  • European Conference on Rare Diseases & Orphan Products

• Research consortia and organizations:

  • Rare Diseases Clinical Research Network

  • NIH Undiagnosed Diseases Network

  • Global Genes Alliance

  • National Organization for Rare Disorders (NORD)

Following these resources will provide comprehensive coverage of advances in basic science, disease mechanisms, and therapeutic approaches for ST3GAL5-related disorders.

What experimental protocols are most reliable for assessing ST3GAL5 function in different model systems?

For reliable assessment of ST3GAL5 function across different model systems, researchers should consider the following experimental protocols:

• In vitro cell culture models:

  • Patient-derived iPSC differentiation into cortical neurons following established protocols

  • Lentiviral or AAV-mediated gene delivery for rescue experiments

  • Ganglioside extraction and thin-layer chromatography or mass spectrometry for profile analysis

• Animal models:

  • Standardized behavioral testing protocols for St3gal5-/- and St3gal5-/-/B4galnt1-/- mice

  • Tissue-specific analysis of ganglioside content

  • Histopathological assessment of brain tissues

  • Vector administration via ICV or i.v. routes at specified doses (e.g., 2 × 10^13 gcs/kg for ICV)

• Molecular and biochemical assays:

  • Western blotting for protein expression analysis

  • RT-PCR or RNA sequencing for gene expression

  • Luciferase reporter assays for promoter activity

  • Electrophoretic mobility shift assays for transcription factor binding

• Functional assays:

  • Enzyme activity assays using fluorescently-labeled substrates

  • Membrane microdomain analysis

  • Electrophysiological recordings in neuronal cultures