Gene Therapy and Autism: Exploring the Current Potential in Monogenic and Polygenic Autism - Introducing Nexus Gene Therapy

 

But, that genetic magic will come with a big price tag!

  

Gene therapy seems to be the way the pharmaceutical industry wants to go when it comes to severe autism. There is a combination of disinterest and dislike when it comes to repurposing cheap old drugs for new uses in the brain. Very high tech, but ultra expensive, gene therapies are attractive to the industry.

As I was writing this post the phase 3 trial for gene therapy for Angelman syndrome was announced to have failed. There were 129 participants, ages 4–17, all with a genetically confirmed full maternal UBE3A deletion (the most common Angelman genotype). The primary endpoint was a change from baseline in the Bayley-4 cognitive raw score at day 338. The secondary endpoint was net response on the Multidomain Responder Index (MDRI), spanning cognition, receptive communication, behavior, gross motor function, and sleep.

So, in this case the placebo was as good as the real gene therapy. This was a trial where all participants had the same single gene autism. Back to the drawing board!

Those 129 had the same affected gene, same broad class of mutation, but not the identical anomaly. Maybe that matters. Maybe the therapy never reached the right neurons. Maybe it just does not work.  

Anyway, back to the post.

 

Could gene therapy eventually be relevant not only to rare single-gene forms of autism, but also to biologically defined subgroups of polygenic or currently idiopathic autism?

For many years, autism research has faced a fundamental problem: we have become increasingly good at finding genes associated with neurodevelopmental disability, but finding a gene is not the same as knowing how to treat it. Gene therapy may eventually change that.

The most obvious strategy is to identify a defective gene and attempt to restore its function. For several genetically defined neurodevelopmental disorders, gene and RNA therapies have already moved beyond theoretical discussion into preclinical and human clinical development. But there may be a bigger—and less obvious—possibility.

What if gene therapy does not always have to target the gene that originally caused the disorder?

What if it could instead target a downstream biological nexus point: a strategically important gene or physiological control system through which the consequences of many different genetic influences converge? That idea could potentially make genetic technologies relevant not only to monogenic disorders, but eventually to some biologically defined forms of polygenic or idiopathic autism.

This does not mean there will be one gene therapy for autism. Probably the opposite. The future may involve a growing toolbox capable of intervening at different points between:

Genetic variation → Altered biology → Physiological dysfunction → Clinical problems

 

First, what do we mean by gene therapy?

Before going further, it helps to understand the basic chain of biology:

DNA → RNA → Protein

• DNA contains genetic instructions.
• RNA carries copied instructions inside cells.
• Proteins perform most functional cellular tasks.

Different therapies can intervene at different points in this chain. They might:

• Alter DNA directly
• Add a functioning gene
• Change how strongly a gene is expressed
• Block a harmful RNA
• Reactivate a silenced gene
• Temporarily provide instructions for making a protein

So gene therapy is not a single technology. It is a family of tools operating at different stages of genetic expression.

 

Autism is not one genetic disease

Autism is extraordinarily heterogeneous. Some people have a relatively well-defined monogenic disorder in which disruption of a single gene has a major effect on neurodevelopment:

• MECP2 — Rett syndrome
• UBE3A — Angelman syndrome
• SHANK3 — Phelan-McDermid syndrome
• TCF4 — Pitt-Hopkins syndrome
• SYNGAP1 and other genetically defined conditions

Autism can be part of the phenotype in these conditions. But these disorders are biologically distinct from autism resulting from the combined influence of many polygenic variants.

For a monogenic disorder, the logic is straightforward:

Identify disrupted gene → Understand biological consequence → Restore or regulate function

For polygenic autism, there is no single defective gene waiting to be repaired. But that does not mean gene therapy will remain irrelevant.

The Obvious Starting Point: Treating the Upstream Genetic Cause

Gene Replacement

If a person has insufficient function of a particular gene, one strategy is delivering a functioning copy using modified viruses, particularly adeno-associated viruses (AAVs).

Conceptually:

Too little functional protein → Provide genetic instructions to make more

However, neurological targets introduce tight constraints. The therapy must reach the correct brain regions, in the correct cells, at precise doses, for the correct duration. Sometimes, too much of the missing protein is dangerous.

 

Rett Syndrome Shows the Problem of Dosage

Rett syndrome is caused by insufficient function of MECP2. The simple solution would seem to be adding MECP2. However:

• Too little MECP2 causes Rett syndrome.
• Too much MECP2 causes severe MECP2 duplication syndrome.

This presents a classic Goldilocks problem: not too little, not too much, but just enough. The challenge is not merely delivering a gene—it is controlling biological expression within narrow safety margins.

 

Gene unsilencing: Using a gene that is already there

Sometimes the required gene is present but silenced. In Angelman syndrome, the paternal copy of UBE3A is silenced in relevant neurons.

Instead of adding an exogenous gene, several RNA-based strategies attempt to reactivate the silent paternal copy already in the cell. This represents a major conceptual pivot: changing the regulation of an existing gene rather than delivering a replacement.

 

The monogenic landscape: Where do we stand today?

While the downstream nexus hypothesis remains an emerging paradigm for polygenic autism, gene-targeted therapies for rare monogenic neurodevelopmental syndromes have transitioned from academic theory into pivotal human clinical trials.

Because these single-gene disorders share significant clinical overlap with autism, their clinical progress serves as the proving ground for delivery systems, dosage control, and regulatory frameworks.

 

Monogenic Target	Therapeutic Strategy	Current Clinical Phase
MECP2 (Rett Syndrome)	AAV9 Gene Replacement + Regulated Dosage Control	Phase 1/2 Registrational (NGN-401, TSHA-102)
UBE3A (Angelman Syndrome)	Antisense Oligonucleotides (ASOs) for Paternal Gene Unsilencing	Phase 3 mixed: GTX-102 Aspire trial missed its primary endpoint (Sept 2026); ION582 in Phase 1/2
TCF4 (Pitt-Hopkins Syndrome)	ICV Delivery AAV Gene Therapy (TCF4 Transgene Replacement)	Early Phase 1/2 (MZ-1866)
SHANK3 (Phelan-McDermid)	AAV Delivery Target Replacement & Small-Molecule Modulators	JAG201 in early Phase 1/2; NNZ-2591 (small molecule) advanced to Phase 3 (Koala study)

 

1. MECP2 — Rett Syndrome (The Dosage Control Benchmark)

• Mechanism: Delivering functional copies of the MECP2 gene via AAV9 vectors.
• Current Status: Advanced Phase 1/2 and registrational studies (e.g., Neurogene’s NGN-401 and TSHA-102).
• Key Translation Insight: Early attempts at unconstrained MECP2 gene replacement carried extreme risks of MECP2 duplication toxicity. Leading clinical programs now incorporate engineered dosage-control platforms (such as built-in microRNA binding sites or regulated promoter systems) designed to restrict protein expression to physiological windows.

 

2. UBE3A — Angelman Syndrome (The Unsilencing Benchmark)

• Mechanism: Intrathecally delivered Antisense Oligonucleotides (ASOs) designed to degrade the UBE3A-ATS transcript, thereby un-silencing the healthy paternal copy of UBE3A in neurons.
• Current Status: Mixed. Ionis’s ION582 remains in Phase 1/2. Ultragenyx’s GTX-102 (apazunersen) reached a fully enrolled, pivotal Phase 3 trial (Aspire), but in September 2026 Ultragenyx announced the trial missed its primary endpoint, showing no significant benefit over sham treatment.
• Key Translation Insight: Unlike one-time viral gene therapy, ASOs require periodic intrathecal administration, offering reversibility and dose titratability. But the Aspire result is a sobering data point in its own right: even a mechanistically well-validated, genetically homogeneous target (a confirmed maternal UBE3A deletion) can fail a rigorously controlled pivotal trial. Ultragenyx has pointed to encouraging long-term open-label extension data (KIK-AS) as reason to keep investigating; whether that reflects a real but harder-to-capture effect, a placebo-sensitive endpoint, or a genuinely ineffective mechanism is still unresolved. Either way, it is a live illustration of the same lesson bumetanide already taught: a plausible downstream target can look strong in early, uncontrolled data and still not clear a controlled, blinded trial.

 

3. TCF4 — Pitt-Hopkins Syndrome (Direct CNS Delivery)

• Mechanism: Direct central nervous system delivery (such as intracerebroventricular injection) of AAV vectors carrying functional TCF4 genetic instructions.
• Current Status: Phase 1/2 early-stage safety and dosing trials (e.g., MZ-1866).
• Key Translation Insight: Because TCF4 is a crucial transcription factor across brain development, achieving widespread CNS biodistribution while avoiding systemic viral exposure is the core technical focus.

 

4. SHANK3 — Phelan-McDermid Syndrome (Synaptic Scaffolding)

• Mechanism: AAV-mediated targeted gene replacement (e.g., JAG201) aimed at restoring synaptic density and post-synaptic scaffolding proteins, paired with small-molecule downstream pathway modulators (e.g., NNZ-2591).
• Current Status: JAG201 (AAV gene replacement) is in early Phase 1/2 cohort dosing. NNZ-2591, a small-molecule downstream modulator rather than a gene therapy, has advanced further — a Phase 2 trial reported symptom improvements, and it is now in a pivotal Phase 3 trial (Koala).
• Key Translation Insight: SHANK3 insufficiency directly impacts synaptic structural integrity. Trials here provide a crucial model for whether structural synaptic restoration in older individuals can yield measurable behavioral or functional gains.

 

What these clinical trials teach us about polygenic applications

1.     Delivery Architecture: The shift away from high-dose IV administration toward intrathecal or intracerebroventricular routes in these monogenic trials directly addresses vector safety concerns, establishing lower-dose CNS administration protocols.

2.     Reversibility vs. Durability: The parallel development of one-time AAVs (Rett) and repeatable ASOs (Angelman) illustrates the trade-off between permanent modification and adjustable maintenance dosing. It is not yet a precedent for which approach works better: GTX-102’s pivotal-trial miss is a reminder that reversibility is a safety and flexibility advantage, not a guarantee of efficacy.

3.     Biomarker Validation: Every monogenic success relies on rigorous molecular and electrophysiological endpoints (such as quantitative EEG patterns or specific protein assays)—reinforcing the argument that downstream polygenic targets will fail without equivalent biomarker stratification.

 

The harder question: What about polygenic autism?

In polygenic autism, correcting every contributing upstream genetic variant is unfeasible:

However, upstream genetic complexity does not necessarily produce equal complexity downstream. Multiple upstream variants may converge on shared biological systems, including:

• Synaptic function and plastic adjustments
• Neuronal excitability and E/I balance
• Gene regulation networks and chromatin remodeling
• Protein translation pathways
• Cellular metabolism and mitochondrial function

 

The central thesis: The gene targeted therapeutically does not necessarily have to be the gene that originally caused the disorder.

 

A Remarkable Experiment in Down Syndrome

The clearest preclinical proof-of-concept for downstream nexus targeting comes from Down syndrome.

Down syndrome involves an extra copy of chromosome 21, altering the dosage of hundreds of genes simultaneously. Correcting the primary chromosomal defect across the brain is currently out of reach.

In 2021, Parrini and colleagues targeted a downstream physiological system: neuronal chloride regulation. Using AAV-mediated, neuron-specific RNA interference, they reduced expression of the NKCC1 chloride transporter in the Ts65Dn mouse model of Down syndrome.

 

The researchers did not correct chromosome 21. They targeted a downstream physiological consequence instead. This experiment provides a concrete model for how complex neurodevelopmental disorders might be approached.

 

NKCC1: An Example of a Therapeutic Nexus

NKCC1 (encoded by SLC12A2) imports chloride into cells:

NKCC1 expression → Intracellular chloride level → Neuronal response to GABA → Network inhibition

 

But didn't Bumetanide fail?

Bumetanide, a small-molecule drug that inhibits NKCC1, was studied as a potential autism treatment. Early pilot studies generated interest, but two large Phase III trials enrolled 211 participants each and found no significant benefit over placebo across the overall unselected ASD study population, leading to early trial termination.

This negative result is important, but comparing systemic bumetanide trials to neuron-specific gene therapy highlights three distinct scientific variables:

Parameter	Systemic Bumetanide Trials	Preclinical AAV-NKCC1 Study
Targeting	Systemic drug with limited brain penetration	Targeted CNS neuronal knockdown
Population	Broad, unselected clinical ASD population	Defined biological model of chloride elevation
Mechanism	Transient pharmacological inhibition	Stable genetic recalibration of expression

 

Broad clinical trials can mask efficacy if a drug is tested across a heterogeneous population where only a fraction of participants possess the targeted biological abnormality. Targeted interventions require verified biomarkers.

 

A new example: Gene editing a downstream metabolic nexus

A striking new example comes from cholesterol rather than autism. In August 2026, researchers reported one-year results from the first-in-human Phase 1a trial of CTX310, a one-time gene therapy treatment designed to switch off ANGPTL3 in liver cells. ANGPTL3 is a regulator of lipid metabolism, and naturally occurring loss-of-function variants in this gene are associated with lifelong reductions in LDL cholesterol and triglycerides. In 15 adults with difficult-to-control lipid disorders, the highest dose of CTX310 produced a mean 52.5% reduction in LDL cholesterol and 47.8% reduction in triglycerides after one year.

The treatment was not correcting the original genetic causes of the patients' lipid disorders; instead, it was deliberately changing a downstream physiological control point. This is conceptually interesting for the hypothesis developed here. It provides a real human example of the principle that a genetic therapy does not necessarily have to repair the original disease-causing mutation: if different upstream causes converge on a measurable biological abnormality, it may sometimes be possible to intervene at the downstream nexus instead.

Of course, CTX310 is a lipid therapy, not an autism treatment, and this small Phase 1 study does not demonstrate that the same strategy will work in autism. But it shows that the broader concept of genetically modifying a downstream physiological regulator rather than correcting the initiating genetic defect has now entered human clinical research

  

Gene therapy does not have to mean permanent DNA editing

"Gene therapy" encompasses tools with varying degrees of reversibility:

  

Controllability as a safety feature

Permanent DNA edits carry risks if the target selection, tissue specificity, or dosing proves suboptimal.

Conversely, reversible tools (such as transient RNA or mRNA therapies) allow dose adjustments, treatment pauses, or modifications over time. For downstream target exploration, controllability is a key safety feature.

 

The economics of gene therapy will shape what gets developed

Monogenic vs. Nexus Economics

• Ultra-rare monogenic targets: Clear molecular mechanisms, but high development costs distributed over small patient populations create commercial challenges.
• Shared downstream targets: If multiple distinct conditions share a downstream biological mechanism, a single intervention could theoretically address a biomarker-defined subgroup spanning several diagnostic categories.

 

 

Durable vs. Repeat-Dose Models

• One-time durable therapies: High upfront costs, long-term regulatory monitoring requirements, and payment structure hurdles for healthcare systems.
• Repeat-dose RNA/mRNA therapies: Distributed costs over time, potential for adjustments, but ongoing administration requirements for patients.

 

Reusable delivery platforms

Developing standardized delivery capsids or lipid nanoparticles capable of carrying different genetic payloads to specific brain cell types could shift the paradigm from one disease, one custom technology toward one platform, multiple biological payloads.

 

Safety: Gene therapy can be dangerous

Gene therapy carries significant physiological risks. In 2025, safety actions were taken following fatal acute liver failure cases in high-dose systemic AAVrh74 trials (Sarepta/Elevidys).

Risks depend heavily on delivery route, vector, and total dose:

• Systemic high-dose IV administration: Requires large viral loads to achieve brain exposure, driving systemic organ exposure and hepatic toxicity.
• Direct CNS-directed administration: Intrathecal or intracerebroventricular delivery targets the central nervous system directly, significantly reducing total vector load and systemic exposure.

 

Evaluating risk

Evaluating risk requires comparing potential treatment adverse events against the natural history of the untreated condition:

 

 

Because autism varies from stable clinical profiles to severe conditions with profound self-injury or treatment-resistant epilepsy, acceptable safety margins will differ depending on individual medical contexts.

 

The translation pathway: A stepwise sequence

Before durable genetic interventions are considered for downstream targets, evidence must be established sequentially:

  

Summary of Evidence Status

Polyvalent mRNA nexus therapy: the equivalent of a "genetic polypill"

If we accept that polygenic autism involves multiple converging biological pathways, targeting a single downstream gene—whether NKCC1, EIF4E, or another physiological node—may rarely capture the full biological picture for a given individual.

This raises a compelling long-term question: Could the clinical principle of a personalized multi-target "Polypill" eventually be realized through programmable RNA platforms?

 

The concept: modular, multi-payload delivery

In immunology, polyvalent vaccines combine distinct antigen sequences into a single formulation to address multiple viral strains simultaneously. A polyvalent mRNA platform for central nervous system (CNS) targets would operate on a similar architecture:

1.     A universal delivery platform: Utilizing advanced delivery vehicles—such as cell-type-specific lipid nanoparticles (LNPs) or engineered viral capsids—designed to cross the blood-brain barrier and target specific neural cells.

2.     Modular payload cocktails: Rather than delivering a single transcript, the platform carries a custom-blended payload ratio tailored to an individual’s specific biological profile:

o    Payload A: Recalibrating neuronal chloride regulation (e.g., modulating NKCC1/KCC2 ratios).

o    Payload B: Normalizing hyperactive translation initiation pathways (e.g., dampening EIF4E or mTOR signaling).

o    Payload C: Supporting mitochondrial cellular bioenergetics or resolving neuroinflammatory responses.

 

The intervention becomes genuinely personalized and pathway-driven, rather than label-based.

 

Key advantages: reversibility and developmental adaptability

Unlike permanent DNA editing, mRNA-based interventions are transient. In complex neurodevelopmental conditions, this transience provides two critical clinical safeguards:

• Age-adaptive formulations: Human brain biology is not static. A young child at age four may require a payload formulation optimized for synaptic maturation and circuit refinement, whereas the same individual at age sixteen might benefit from a revised ratio targeting network excitability or cellular metabolism.
• Titratability and safety: If an individual experiences an adverse response or if an objective biomarker fails to shift as predicted, the dosage of a specific payload component can be adjusted, swapped, or stopped entirely in subsequent administrations. Controllability serves as a primary safety mechanism.

 

A long-term horizon, not an imminent reality

To be clear polyvalent mRNA therapy represents a long-term conceptual horizon—a vision spanning decades—rather than an imminent treatment. Moving from theory to practice requires overcoming profound technical hurdles:

• Validating reliable, non-invasive biomarkers capable of measuring downstream pathway activity in living human brain tissue.
• Achieving cell-type specificity (e.g., excitatory neurons vs. inhibitory interneurons vs. astrocytes) without off-target toxicity.
• Determining precise therapeutic windows for multi-target RNA dosing in developing neural circuits.

Yet, while the engineering required to deliver multi-target brain therapies remains futuristic, the underlying principle is already relevant today, moving away from broad diagnostic categories toward precise, biomarker-guided combinations that address an individual’s actual biology.

Betaine (TMG) and Gene Therapy as potential alternatives to Bumetanide Treatment in Autism?

Betaine (also known as TMG, or trimethylglycine) is a methyl derivative of glycine, first isolated from sugar beet and hence its name.

Today’s post was prompted by our reader, and Covid home-school instructor, AJ.  He raised the question of whether betaine can be used like Bumetanide to normalize chloride levels in neurons.

I am combing this idea with news from Genoa in Italy, where they have developed gene therapy as an alternative to Bumetanide and in their words :-

“This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.”

The interesting thing is that neither of these ideas come from autism research.  The idea to use Betaine was stumbled upon and was then written up in a Norwegian case study about Creatine transporter deficiency.  The Italians are trying to improve cognition in brain disorders and their model of choice was Down syndrome. 

As we have seen time and again, elevated chloride within neurons is a common feature of many types of brain disorders from some idiopathic autism, to Down syndrome, to adult conditions such as Parkinson’s disease.  Today we learn that it is may well be a feature of Creatine Transporter Deficiency.

I have been rather wary of writing about any kind of gene therapy, because it seemed either too far ahead of its time, or just absurdly expensive.  There are some new $1+ million treatments.

This may be about to change given that the Biontech (AKA Pfizer vaccine), Moderna, Janssen (Johnson & Johnson) and Oxford AstraZeneca vaccines for Covid 19 are all based on gene therapy.

The Biontech people are really clever and were already trying to treat various kinds of cancer and other condition using gene therapy, before they developed their highly successful Covid vaccine.

The Italians in Genoa used an adeno-associated virus (AAV)-mediated RNA interference (RNAi) to target and reduce neuronal NKCC1 expression, rescue neuronal Cl-  homeostasis, GABAergic transmission, and cognitive deficits.   The benefit was still there 6 months after the injection.

Don’t worry if the above paragraph makes little sense. Just read on.

The same type of adeno-associated virus (AAV) vector is the platform for gene therapy delivery used in the Astra Zeneca, Janssen and the Russian Sputnik covid vaccines.

The virus is just the delivery system (vector) to get some genetic code into cells.

The Oxford-AstraZeneca COVID-19 vaccine uses a chimpanzee adenoviral vector. It delivers the gene that encodes the SARS-CoV-2 spike protein, to our cells.  Our cells then transcribe this gene into messenger RNA, or mRNA, which in turn prompts our cellular machine to make the spike protein in the main body of the cell. The mRNA molecule behaves essentially like a recipe.  Then our cells present the spike protein on the cell surface, prompting our immune system to make antibodies and mount T cell responses.

Biontech and Moderna are pioneers of mRNA vaccines, which bypass one step in the above process. They do not require our cells to make the messenger RNA, or mRNA.  They have already made it for you.

 

Gene therapy for autism?

Single gene autisms are all potential candidates for gene therapy.

The problem is that most autism and all Down syndrome is polygenic, there can be hundreds of miss-expressed genes.

But the researchers in Italy show us that even polygenic autism and Down syndrome can benefit from therapy targeting a single gene.  You just have to select the right one.

The problem is the price. Covid vaccines are made in huge quantities and are cheap.

Customized gene therapy is ultra expensive, in part because each therapy has to be approved individually.

 

An NKCC1 Gene Therapy?

The Italians have already made the NKCC1 Gene Therapy.  The question is will it ever going be available to humans with Down Syndrome, Autism or even Parkinson’s disease?

Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome

A common feature of diverse brain disorders, is the alteration of GABA-mediated inhibition due to aberrant intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of GABA-mediated inhibition and neuronal network dynamics in vitro and ex vivo. Importantly, AAV-mediated neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice, and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

 

This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.

This highlights a causative role of NKCC1 upregulation in learning and memory deficits in adult Ts65Dn mice, thus also validating brain NKCC1 as a target for ameliorating cognitive disabilities in DS. Furthermore, our neuro-specific knockdown approach points to neurons as major players in the NKCC1- dependent cognitive impairment in DS mice. Nevertheless, we cannot exclude that other cell types which also express NKCC1 (e.g. glial cells) could still play a role in the overall cognitive impairment that characterizes DS.

Despite the very large and fast-increasing literature both on animal models and patients indicating positive outcomes upon bumetanide treatment, there is not yet a strong demonstrated direct link between NKCC1 inhibition, restoration of Cl- homeostasis and full GABAergic inhibitory signaling, and rescue of brain deficits.  Moreover, bumetanide has strong diuretic activity, triggering ionic imbalance, and potential ototoxicity 25,26.  This hampers its use for clinical applications in lifelong treatments4,27 and may strongly jeopardize treatment compliance along years of treatment.  Moreover, bumetanide was given systemically in most studies, and the suboptimal brain pharmacokinetic profile of the drug28 raises questions on its mechanism of action29.  Here, we demonstrate that adeno-associated virus (AAV)-mediated RNA interference (RNAi) to target (and reduce) neuronal NKCC1 expression rescues neuronal Cl- homeostasis, GABAergic transmission, and cognitive deficits in the Ts65Dn mouse model of Down syndrome. This sets the stage for the development of a gene therapy approach to overcome the shortcomings of bumetanide treatment.

 

“Thus, our results indicate the efficacy of long-term AAV9-mediated neuro-specific NKCC1 knockdown in rescuing cognitive deficits in Ts65Dn mice.”

 

“Besides establishing a causal link between NKCC1 upregulation and cognitive impairment in DS, our data also provide a proof-of-concept for a neuro-specific RNAi gene therapy approach to restore hippocampus-dependent cognitive behaviors in adult animals specifically in the brain, and without affecting peripheral organs (e.g., the kidney). This is particularly relevant in the context of the current clinical trials repurposing the strong diuretic bumetanide to treat brain disorders with impaired chloride homeostasis3.  Importantly, we achieved a comparable degree of long-term cognitive rescue with two different amiR sequences against NKCC1, underlining the specificity of our approach.”

  

Gone Fishing

If a trip to Italy for gene therapy is not realistic, this takes us back to AJ’s idea, which is to use Betaine.  The correct version is TMG or glycine betaine, and confusingly not Betaine HCl.

Fish love the taste of betaine.

Betaine was first isolated from sugar beet.

I recall from my time at the sugar factory, when I was 18, that once you have sliced up the sugar beet and extracted as much sugar as possible you are left with the pulp.  This pulp is dried, molasses is added back and then it is made into pellets.  The pellets are fed to cattle and horses.  They taste pretty bad in my opinion.

To humans it tastes bad because of the beet molasses by-product.

The molasses by-product from sugar cane tastes great to humans.  That is why they make rum in the Caribbean, and not in England or Canada.

Brown sugar from a sugar beet factory is made by adding sugar cane molasses to white sugar from beet.  It is a cheat really.

Cows love sugar beet by-products.

It turns out that fish love betaine HCl.

Betaine HCl is an excellent natural attractor that stimulates a strong, prolonged feeding response from carp and many other coarse fish.

Betaine HCl is now used to induce feeding in the fish farming industry

As our reader Tyler has highlighted, Betaine HCl, that fish like and is available is a cheap supplement is not the same as the Betaine used in the medical case study. Confusingly, the original Betaine (TMG, or called glycine betaine) gave way to a class of compounds all called betaines. One of these betaines is betaine HCL.

In most cases, in the medical literature when they refer to Betaine, they mean glycine betaine, also known as TMG.

Betaine HCl is used to increase acidity in your stomach. The effect of betaine compounds other than glycine betaine/TMG on NKCC1 is unknown.

Glycine Betaine (TMG) and NKCC1

It seems that betaine reduces your level of NKCC1 RNA. 

In your DNA are the instructions to make the NKCC1 transporter. To go from these instructions to actually making the transporters you need RNA.

In some autism there are too many NKCC1 transporters, so put simply there was too much NKCC1 RNA. So, if you can find a substance that reduces NKCC1 RNA, you might well solve the problem.

The caveat is that the substance must not also increase KCC2 RNA.  This appears to be what taurine does.

Here, finally, is AJ’s paper:

Treatment experience in two adults with creatine transporter deficiency

Background

Creatine transporter deficiency (CTD) is an X-linked form of intellectual disability (ID) caused by SCL6A8 mutations. Limited information exists on the adult course of CTD, and there are no treatment studies in adults.

Methods

We report two half-brothers with CTD, 36 and 31 years at intervention start. Their clinical phenotypes were consistent with CTD, and intervention was indicated because of progressive disease course, with increased difficulties speaking, walking and eating, resulting in fatigue, and malnutrition. We therefore performed treatment trials with arginine, glycine and a proprietary product containing creatine and betaine, and then a trial supplementing with betaine alone. Results In the older patient, glycine and arginine were accompanied by adverse effects, while betaine containing proprietary product gave improved balance, speech and feeding. When supplementation stopped, his condition deteriorated, and improved again after starting betaine supplement. Betaine supplementation was also beneficial in the younger patient, reducing his exhaustion, feeding difficulties and weight loss, making him able to resume his protected work.

Discussion & conclusion

We report for the first time that betaine supplement was well tolerated and efficient in adults with CTD, while arginine and/or glycine were accompanied by side effects. Thus, betaine is potentially a new useful treatment for CTD patients. We discuss possible underlying treatment mechanisms. Betaine has been reported to have antagonistic effect on NKCC1 channels, a mechanism shared with bumetanide, a medication with promising results in both in autism and epilepsy. Further studies of betaine's effects in well-designed studies are warranted.

 

The mechanism of betaine’s assumed favorable effect is unknown. We do not know whether betaine influences the cell creatine content in itself or its effects are more aspesific. However, we would like to present some hypotheses. First, betaine may have effect in CTD by modulating GABA-transmission. Betaine has been reported to have an antagonistic effect on NKCC1 channels, which also influences GABAergic neurotransmission. Inhibiting NKCC1 is a mechanism shared with bumetanide, a well-known diuretic medication that in recent years has been found to influence GABAergic transmission, and thereby it has been found promising in treatment of several brain conditions, including autism, and epilepsy. NKCC1 inhibition by bumetanide has also been tried with success in other rare neurodevelopmental disorders fragile X syndrome and tuberous sclerosis. Second, betaine’s properties as an osmolyte may be of importance, as betaine has similarities with creatine in being an osmolyte. Osmotic properties are thought to be one of the central mechanism behind bumetanide’s efficacy in treating brain disorders. Thus, it could be speculated that the lack of intracellular creatine in CTD may result in inefficient osmolyte regulation, and that betaine supplementation replaces the lacking creatine and thereby improves the neuronal adaption to salinity changes, edema or cellular dehydration. Betaine has osmolyte properties that even makes it act as a “chemical chaperone” increasing the stability of cell and membrane proteins. Fourth, it is possible that betaine has some effect through modifying methylation. Methylation of GAA by GAMT to form creatine is a rate-limiting step in the creatine synthesis by neurons. Betaine could stimulate this by donating methyl groups to SAMe, which donates a methyl group to GAA to form creatine. This might reduce the burden when body demands more methyl groups for creatine synthesis. Similar mechanisms may be responsible for a beneficial effect of both betaine and s-adenosyl methionine (SAMe). However, as creatine and GAA share the same transporter, one would not expect GAA to enter the GAMTexpressing cells in patients suffering from CTD. Still, it cannot be excluded that there is some rest function in the creatine transporter, and that increased endogenous synthesis improves the condition slightly. Furthermore, it is possible that CTD increases the need for methylation agents in general, as creatine supplementation has been found to reduce the need for other methylation agents [34]. Thus, it is likely that betaine may have a positive effect in CTD by improving methylation capacity for other reactions than those directly involved in creatine production. Betaine’s effect on muscle may be also of importance, as animal studies have shown that muscles growth improves with betaine [35], which potentially could have had a positive impact on our patients fatigue and weight loss. To summarize, betaine has several properties that make it likely that it will have a beneficial effect in CTD, especially the properties as an osmolyte, a down regulator of the NKCC1 channel and an influencer of GABAergic transmission. These properties are similar to the properties of bumetanide, a promising new medication for treatment of autism and epilepsy, which are common symptoms of CTD. Further research is needed, however, to elucidate the role of betaine in CTD.

If you read the detail of the old paper that is referred to in the above paper, you see that betaine is not blocking the NKCC1 channels as suggested, but it seems to be reducing the number of them.  The net effect may be the same, but the process is very different.

 

Expression and regulation of the Na+/K+/2Cl− cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells

The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in different liver cell types. NKCC1 was found in rat liver parenchymal and sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1 expression in rat hepatic stellate cells increased during culture-induced transformation in the myofibroblast-like phenotype. NKCC1 inhibition by bumetanide increased α₁-smooth muscle actin expression in 2-day-cultured hepatic stellate cells but was without effect on basal and platelet-derived-growth-factor-induced proliferation of the 14-day-old cells. In perfused rat liver the NKCC1 made a major contribution to volume-regulatory K⁺ uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by elevation of extracellular NaCl or raffinose concentration but not hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA and protein expression. This was antagonized by the compatible organic osmolytes betaine or taurine. The data suggest a role of NKCC1 in stellate cell transformation, hepatic volume regulation, and long-term adaption to dehydrating conditions.

 

Aha!  Glycine Betaine and Taurine – not so fast 

You have to check the effect on both NKCC1 and KCC2.  One lets chloride into neurons and the lets it out.  You want to block NKCC1 and not KCC2, otherwise you undo all the good you have done.

Both glycine betaine (TMG) and taurine are already used as autism supplements at low doses.  The paper below suggest that Taurine is not a good idea for people with high levels of chloride within neurons.

 

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.

 

So, it is likely only Glycine Betaine (TMG) may be of potential benefit, in the case of lowering chloride.

 

Glycine Betaine in the broader research

 

Betaine in Inflammation: Mechanistic Aspects and Applications

Betaine is known as trimethylglycine and is widely distributed in animals, plants, and microorganisms. Betaine is known to function physiologically as an important osmoprotectant and methyl group donor. Accumulating evidence has shown that betaine has anti-inflammatory functions in numerous diseases. Mechanistically, betaine ameliorates sulfur amino acid metabolism against oxidative stress, inhibits nuclear factor-κB activity and NLRP3 inflammasome activation, regulates energy metabolism, and mitigates endoplasmic reticulum stress and apoptosis. Consequently, betaine has beneficial actions in several human diseases, such as obesity, diabetes, cancer, and Alzheimer’s disease.

 

Betaine is a stable and nontoxic natural substance. Because it looks like a glycine with three extra methyl groups, betaine is also called trimethylglycine . In addition, betaine has a zwitterionic quaternary ammonium form [(CH3)3N+ CH2COO−] (Figure 1). In the nineteenth century, betaine was first identified in the plant Beta vulgaris. It was then found at high concentrations in several other organisms, including wheat bran, wheat germ, spinach, beets, microorganisms, and aquatic invertebrates. Dietary betaine intake plays a decisive role in the betaine content of the body. Betaine is safe at a daily intake of 9–15 g for human and distributes primarily to the kidneys, liver, and brain. The accurate amount of betaine intake generally relies on its various sources and cooking methods. Besides dietary intake, betaine can be synthesized from choline in the body. Studies report that high concentrations of betaine in human and animal neonates indicate the effectiveness of this synthetic mechanism.

  

Boosting amino acid derivative may be a treatment for schizophrenia

Many psychiatric drugs act on the receptors or transporters of certain neurotransmitters in the brain. However, there is a great need for alternatives, and research is looking at other targets along the brain's metabolic pathways. Lack of glycine betaine contributes to brain pathology in schizophrenia, and new research shows that betaine supplementation can counteract psychiatric symptoms in mice.

 

 

Supplement treats schizophrenia in mice, restores healthy “dance” and structure of neurons Repurposed drug works by building cells’ skeleton and transportation network

 

 

Conclusion

Early on in the Covid saga, I saw interviews with both the Moderna researchers and the Oxford (AstraZeneca) researchers. Both claimed that they designed their vaccines over a weekend.  This was made possible by the Chinese releasing the DNA code of the virus.

When you think about gene therapy for autism and Down syndrome, the same likely applies; much could be achieved over a weekend.

The expensive and time-consuming part is the testing and approval process.

In the Covid pandemic the approval process was modified to allow for emergency use.  Perhaps this should also be the case for all gene therapies?

What use is a $2 million therapy for autism or Down syndrome?

In theory, if you gave your gene therapy prior to birth or shortly thereafter, it might be fully curative.  Realistically, by the time you get the therapy it is just going to be beneficial and you will still need other ongoing therapies.

Note that gene therapy normally applies to just one gene.  In Down syndrome people have a third copy of all, or just part, of Chromosome 21.  This results directly in the miss-expression of hundreds of genes from that chromosome.

The gene that encodes NKCC1 is on Chromosome 5, which has nothing directly to do with Down syndrome.

The NKCC1 transporter is over-expressed in Down syndrome as a down stream consequence of the disorder. It is caused by the “faulty GABA switch”, referred to in earlier posts.

The Italian gene therapy to lower chloride in neurons and so raise cognition, has numerous applications, in people currently of all ages, so there is a big potential market.

Why not gene therapy for all single gene autisms?  It could be a highly productive use of the researcher’s weekends, for a year or two.

The issue is who would pay for the $20 to $30 million approval process, for each gene?

Maybe some of the billions in profit from clever Covid vaccines could be used for pro bono gene therapy?  Highly unlikely.

Biontech, who are the brains behind the Pfizer vaccine, do have plans to develop gene therapy for other medical conditions.  I think these will be ultra expensive,

That brings me back to Glycine Betaine (TMG), is 10g a day of this supplement really going to reduce the expression of NKCC1 transporters in neurons and so lower chloride within neurons?  It seems to work in creatine transporter deficiency, is all we can say.  

Glycine betaine, at much lower doses, has been used by DAN and now MAPS doctors for decades. They use it as a “methyl-donor”.  There is a combination of real science and hocus-pocus surrounding DNA methylation. 

 DNA Methylation and Susceptibility to Autism Spectrum Disorder