Editor’s Note: This story ran in DDN’s November print issue, which was mailed to DDN print subscribers on November 1, 2024. On November 11, Neurogene — one of the companies developing a Rett syndrome gene therapy featured in this story — announced that one patient in their clinical trial’s high-dose cohort experienced a severe adverse event to the gene therapy. On November 18, the company stated that the patient was in critical condition and that Neurogene had halted the high-dose arm of the trial. In a November 21 filing with the Securities and Exchange Commission, Neurogene reported that the patient who was previously in critical condition has died. The low-dose arm of the trial will continue.

A girl who was once a happy toddler, babbling a few words and starting to speak in short phrases, stops speaking one day. Another may soon have a hard time gripping a toy or begin wringing her hands and walking on her tiptoes. 

Through genetic testing, many of the young girls with these symptoms will receive a diagnosis of Rett syndrome. 

“This disease affects every part of the body,” said Sukumar Nagendran, who is the President and Head of Research and Development at the biotech company Taysha Gene Therapies. “Regardless of mild to severe, it completely disrupts family's lives.”

Rett syndrome is a neurodevelopmental disorder that arises from mutations in the gene methyl-CpG-binding protein 2 (MECP2) (1). Because MECP2 is present on the X chromosome, the disease most commonly affects girls. Due to X chromosome inactivation, Rett syndrome is a mosaic disease: Some cells will express the X chromosome with the healthy copy of MECP2, while other cells will express the mutant version.

In 2023, the Food and Drug Administration approved the drug trofinetide for Rett syndrome, but it only improves some symptoms of the condition. It doesn’t treat the root cause: loss of MECP2 (2).

For scientists like Neurogene founder and Chief Executive Officer, Rachel McMinn, however, the treatment strategy for Rett syndrome was obvious. In 2017, she read about a gene replacement therapy for kids with the lethal disease spinal muscular atrophy (3). Knowing that a single gene caused Rett syndrome, she put two and two together.

“It's this crystallizing moment of like, gene therapy is going to be a really important treatment modality,” she said. “I read that paper in November, and in January, I founded the company.”

After tackling challenges in expressing just the right amount of MECP2 in neurons as well as ensuring the therapy reached as many neurons as possible, two companies — Neurogene and Taysha Gene Therapies — now have gene therapies for Rett syndrome in Phase 1/2 clinical trials. With positive safety data in hand, they plan to release preliminary efficacy results in late 2024 and the first half of 2025, respectively. They hope that the results will point to MECP2 gene therapy as a strategy to strike Rett syndrome at its source.

MECP2 heals the brain

When University of Edinburgh geneticist Adrian Bird discovered the MECP2 gene in 1992, he and his colleagues had no idea that it caused Rett syndrome (4).

“We wanted to try to understand the function of DNA methylation,” he said. “So, we looked for proteins that might be able to tell whether DNA was methylated or not, and MECP2 came up as a protein that bound only when DNA was methylated.”

Just a few years later, though, Huda Zoghbi and her team at Baylor College of Medicine mapped mutations in multiple patients with Rett syndrome to the MECP2 gene (1).

“The Zoghbi discovery meant that we had a second reason for being interested, and that was a possibility of therapy,” Bird said.

He and his group developed the first animal model lacking MECP2, which exhibited similar neurological symptoms as girls with Rett syndrome (5). They wondered if it might be possible to reverse these symptoms by giving the mice a healthy copy of MECP2. To help his team do that, Bird contacted Stuart Cobb, a neuroscientist also at the University of Edinburgh, to collaborate.

“He's a more molecular person, so he'd reached out to me to bring some neuroscience into the project,” said Cobb. “At the time, Rett syndrome and neurodevelopmental disorders in general were considered basically untreatable because the view was that the nervous system had developed abnormally, and there wasn't much you could do about that. So, we developed an experiment to test that.”

They started with female mice that were heterozygous for loss of MECP2, the same genetic background as girls with Rett syndrome. When the mice began to show symptoms — such as clasping their hindlimbs when picked up, irregular breathing, and lack of movement — the researchers induced expression of MECP2 and watched as the mice now ran around their cages, their Rett syndrome symptoms gone (6).

“Nobody expected it — including us — because usually brain things are thought to be irrevocable,” said Bird. “But in this case, that isn't true.”

The effects of adding back MECP2 lent support to the gene therapy’s potential for Rett syndrome. But as researchers began developing ways to deliver MECP2 to animal models and eventually people, they realized that while too little MECP2 expression leads to Rett syndrome, too much is not good either.

Building a genetic thermostat

Classical gene therapies take a healthy copy of a gene and deliver it to a cell, typically via an adeno-associated viral (AAV) vector. Normally, the expression of the gene will be under the control of a strong promoter, leading to high expression of the gene that’s being delivered.

“Gene therapy is a bit of a blunderbuss approach,” said Bird. “It seems high tech, but actually, it's pretty crude because you're going in, putting in a good gene on top of a bad gene.”

This becomes a problem for treating Rett syndrome in particular because MECP2 expression must be precise. Too little MECP2 leads to Rett syndrome, but too much causes a condition called MECP2 duplication syndrome, which is a severe neurodevelopmental disorder that is as serious as Rett syndrome.

“It kills mice even faster than when they don't even have any MECP2 at all,” said McMinn. So, when it came to designing a gene therapy for Rett syndrome, “we had to build in almost like a genetic thermostat.”

To do that, McMinn reached out to Cobb as one of the leaders in the Rett syndrome field. 

Gene therapy is a bit of a blunderbuss approach. It seems high tech, but actually, it's pretty crude because you're going in, putting in a good gene on top of a bad gene. 
- Adrian Bird, University of Edinburgh

“Within no time, she was flying across to Europe to visit me in Edinburgh, and we spent two days talking solid science,” Cobb said, who is now Neurogene’s Chief Scientific Officer in addition to holding his academic position.

At first, he and his team experimented with changing the promoter driving MECP2 expression or lowering the dose of the AAV9-based gene therapy, but they couldn’t find a combination that was both effective and safe. After going back to the drawing board, Cobb and his team developed a microRNA-based regulatory system that the Neurogene team named EXACT. The EXACT technology works by expressing both a healthy copy of MECP2 in tandem with a microRNA that specifically inhibits that MECP2 transgene. Every time a cell expresses the healthy MECP2 transgene, it will also express the microRNA regulator, thus preventing MECP2 expression from getting too high. Neurogene’s gene therapy for Rett syndrome that uses this EXACT regulatory technology is called NGN-401.

“Some people ask, ‘How do you know what [MECP2] levels are the right levels to achieve?’ And we didn't,” said McMinn. “We just created approximately a dozen different versions of NGN-401, if you will, modifying one genetic element at a time, and then we just selected the product that had the best profile,” meaning that it maintained MECP2 levels within a safe and effective dose. 

The researchers at Taysha Gene Therapies developed a similar approach to help regulate MECP2 expression in their AAV9-based gene therapy called TSHA-102. They deliver both a mini-version of the healthy MECP2 gene along with a microRNA Responsive Auto-Regulatory Element (miRARE), which is a sequence that contains binding sites for microRNAs that regulate MECP2 expression (7). The smaller size of the mini-MECP2 gene construct, which improves symptoms in Rett syndrome animal models just as well as the full-length gene, gives the researchers more room in the AAV9 vector to add these regulatory elements (8).

“Our technology is actually very sensitive to both the endogenous MECP2 and its own MECP2 which is exogenous,” said Nagendran. So, if MECP2 expression levels get too high, “there’s a microRNA that comes and attaches itself to the transcript, plays a role in cycling [it] out into the cytoplasm, and reduces it,” he added.

Neurogene and Taysha Gene Therapies also differ in how they deliver their Rett syndrome gene therapies to reach patients’ brains. In a nonhuman primate study, the Neurogene team found that their therapy had the best distribution in the brain when they administered it through an intracerebroventricular (ICV) injection, which puts the therapy directly into the cerebrospinal fluid (CSF) in the ventricles of the brain (9).

“To develop an effective treatment, we've got to get it to enough cells in the brain, and then on top of that, we need to make sure that the procedure itself is not so overly complicated that it can never be scaled up and used,” said McMinn. 

The team at Taysha Gene Therapies, however, delivers TSHA-102 via a lumbar puncture, which puts the therapy into the CSF at the base of the spine where it then travels to the brain. While both ICV injections and lumbar punctures are standard neurosurgical procedures, lumbar punctures are more common.

For Bird, he is glad to see that there are two different approaches that aim to treat Rett syndrome in the clinic, and he is eager to learn more about how each of the companies’ Phase 1/2 trials are going.

“Both Neurogene and Taysha have intimated through press releases that there is no safety issue, so that's already clear. But what everybody wants, of course, is efficacy, and that's expecting a lot with the first safety dose,” he said.

Frankly, I'm blown away as a physician seeing what I'm seeing in a very complex disease, but I think we need time. We need to collect all the data, analyze it. 
- Sukumar Nagendran, Taysha Gene Therapies

Currently, Taysha Gene Therapies has two ongoing trials testing TSHA-102: one in pediatric Rett syndrome patients and one in adolescents and adults. They plan to release data from those studies in the first half of 2025. From what Nagendran has seen so far, he’s excited for what’s to come.

“Frankly, I'm blown away as a physician seeing what I'm seeing in a very complex disease, but I think we need time. We need to collect all the data, analyze it,” he said.

McMinn echoed this sentiment for Neurogene’s trial for NGN-401 in young girls with Rett syndrome.

“Things are going very well. We're very excited,” she said. Neurogene plans to release the first data from their trial in the fourth quarter of 2024. “To provide that hope and the prospect of a potential treatment to modify the disease course,” McMinn said, “that's really what drives me every day.”

With two promising gene therapies in the clinic, people with Rett syndrome are closer than ever before to having a treatment that can potentially reverse the cause of their condition. And the researchers working on these therapies can’t wait to have something that can improve these patients’ lives.

Nagendran said, “I hope all of us who are trying to develop medicines, gene therapies, whatever, for these patients succeed and we make a difference.”

References

1. Amir, R.E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet  23, 185-188 (1999).
2. Furqan, M. Trofinetide—a new chapter in rett syndrome’s treatment. Front Pharmacol  14, 1284035 (2023).
3. Mendell, J.R. et al. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N Engl J Med  377, 1713-1722 (2017).
4. Lewis, J.D. et al. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to Methylated DNA. Cell   69, 905-914 (1992).
5. Guy, J. et al. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet   27, 322-326 (2001). 
6. Guy, J. et al. Reversal of neurological defects in a mouse model of Rett syndrome. Science   315, 1143-1147 (2007).
7. Sinnett, S.E. et al. Engineered microRNA-based regulatory element permits safe high-dose miniMECP2 gene therapy in Rett mice. Brain  144, 3005–3019 (2021). 
8. Tillotson, R. et al. Radically truncated MeCP2 rescues Rett syndrome-like neurological defects. Nature   550, 398–401 (2017). 
9. Daily, J.L., Burstein, S.R., Keiser, N.W., & Cobb, S.R. Abstract 347. A Non-Human Primate Biodistribution Study Comparing Multiple Routes of Administration of AAV9. Mol Ther   29, 171-172 (2021).