Courtney S. Ambrose

The dream of curing genetic disorders has been a persistent but elusive goal, even before the human genome was mapped. Once mapping of the human genome was complete in 2001, an entirely new avenue of potential treatments and cures for genetic diseases and disorders was opened.^1,2

The disorders best suited for targeted gene therapy are monogenic; however, tools and delivery methods for editing the human genome were limited and difficult to apply, until the advent of the CRISPR system in 2012.³ CRISPR (an acronym of clustered regularly interspaced short palindromic repeats) has changed the way in which gene therapy strategies are pursued, with dozens of companies leveraging a variety of platforms to create potentially life-changing therapies for devastating rare diseases and disorders.

One of the rare monogenic disorders that is embracing multiple gene therapy approaches is Rett syndrome, a rare, debilitating neurodevelopmental disorder. In this review, we explore the molecular cause of Rett syndrome, disease presentation, current treatments, ongoing clinical trials, and therapies that are on the horizon.
 

Underlying molecular cause

Rett syndrome is caused by mutations in, or the absence of, the MECP2 gene, which produces methyl-CpG binding protein 2 (MECP2). The syndrome was first described clinically in 1954 by the Austrian physician Andreas Rett; it would take until 1982 before the disorder was officially named, eponymously, in a seminal paper by Hagberg.⁴ After Hagberg’s characterization, Rett syndrome became the predominant global clinical diagnosis identified among cognitively impaired females, with an incidence of 1 in every 10,000 to 15,000.⁴

In 1999, mutations in, and deletions of, MECP2 were identified as the cause of Rett syndrome.^4,5 MECP2 is located on the X chromosome, in the Xq28 region, making Rett syndrome an X-linked dominant disorder.⁶ Rett syndrome is seen predominantly in females who are mosaic for mutant or deleted MECP2. Random X chromosome inactivation results in some cells expressing the mutant MECP2 allele and other cells expressing the normal functioning MECP2 allele; the percentage of cells expressing the normal allele correlates with the degree of syndrome severity.^7-9

The incidence of Rett syndrome is much lower in males, in whom the syndrome was originally thought to be lethal; many observed male cases are either mosaic or occur in XXY males.^10,11

Approximately 95% of cases of Rett syndrome are due to de novo mutations in MECP2, with a handful of specific mutations and large deletions accounting for more than 85% of cases.¹² The fact that Rett syndrome is monogenic and most cases are caused by, in total, only a handful of mutations or deletions makes the syndrome a promising candidate for gene therapy.

At the molecular level, it has been observed that the MECP2 mutations of Rett syndrome lead to loss of gene function, thus disrupting the ability of the MECP2 nuclear protein to regulate global gene transcription through its binding to methylated DNA sites.¹² A large percentage of these missense and nonsense mutations lead to a truncated or nonfunctional protein.¹²

One of the ways in which MECP2 regulates transcription is as a component of heterochromatin condensates and by separation of heterochromatin and euchromatin.^13-15 It has been observed that the cells of Rett syndrome patients have an altered chromatin state, potentially contributing to transcriptional dysregulation.^16,17 Several mutations observed in Rett syndrome patients occur in crucial domains for heterochromatin condensate formation, which helps explain this cellular phenotype.¹³ Introduction of a engineered “mini” MECP2 in a murine model of Rett syndrome has resulted in partial rescue of heterochromatin condensate formation and transcriptional regulation – fostering the hypothesis that correcting those genetic changes could lead to a potential therapy.¹⁸

Beyond the role of MECP2 in heterochromatin condensate formation, the gene interacts with more than 40 proteins that have diverse roles in cellular function, epigenetic modulation, and neuronal development. This volume of interactions contributes to MECP2 being a global gene regulatory protein that has far-reaching effects on transcriptional regulation across the genome.^19-22

Epigenetic dysregulation has been associated with neurodevelopmental and neuropsychiatric disorders.²³ Both insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor are transcriptional targets of MECP2, and are involved in neuronal differentiation, synaptic function, and neurite outgrowth.¹² This helps explain the neurodevelopmental phenotypes observed in MECP2-mutated patients.

Notably, although Rett syndrome patients experience neurodevelopmental phenotypes at the cellular level, neuronal death is not readily observed. That observation provides hope that an interventional therapy after onset of symptoms might still be of benefit.
 

 

Presentation

Early neurotypical development. A hallmark of Rett syndrome is neurotypical physical and mental development until 6 to 24 months of age.

Stagnation is the first stage of the syndrome, involving a small but rapid decline in habitual milestones, such motor and language skills.¹² Subtle signs, such as microcephaly and hypotonia, can also arise at this time but might be missed.²⁴

Rapid regression follows stagnation. Speech and motor delays and impaired gait and breathing occur;^12,25 purposeful hand skills are lost, replaced by repetitive hand-wringing movements that are a hallmark of the syndrome.^12,24 Seizures are observed; they become more common during the next stage.¹²

Plateau. Language advances can be observed, but further deficits are seen in motor skills and hand coordination.¹²

Late motor deterioration stage. Late physical deficits develop, leading to lifelong impairments. The physical deficits observed are the result of severe muscle weakness, usually resulting in wheelchair dependency.¹²

Plateau. Patients then reach a second plateau. Regression stops; deficient physical and cognitive states stabilize and are maintained.²⁵

At all stages of Rett syndrome, the following are observed:

• Gastrointestinal problems.
• Sleep disturbances.
• Abnormal cardiorespiratory coupling.
• Greater-than-expected mortality.¹²

Final regression. The patient is fully dependent for the rest of their lifespan, partially due to seizure activity.^26,27
 

A life-changing diagnosis

A diagnosis of Rett syndrome is life-changing for a patient’s family; access to supportive groups of other patients and their families is extremely beneficial. Two helpful organizations – the Rett Syndrome Research Trust²⁸ and International Rett Syndrome Foundation,²⁹ – offer patient support and community and fund research.

Because X chromosome inactivation is random in Rett syndrome, the individual patient can present with a wide variety of phenotypic combinations – making the patient, and their needs, unique.¹² During stages of regression, patients often experience emotional dysregulation and anxiety, which is attributable to their increasing physical difficulties.³⁰ They often exhibit combinations of uncontrolled movements, including repetitive rocking, scratching, and self-injurious behavior.³⁰ For most, regression subsides after the first 5 years of alternating development and regression; after that, their ultimate symptoms persist for life.²⁵

As patients mature, they need to be monitored for proper nutrition and scoliosis.²⁵ As adults, they are at risk of pneumonia, respiratory distress, status epilepticus, osteopenia, and lack of adequate food or water because of impaired ability to feed.²⁵

The lifespan of Rett syndrome patients has increased, thanks to improvements in health care, advances in technology, and early genetic testing, which allows for earlier diagnosis, intervention, and management of symptoms.
 

Current treatments

When a female patient presents with regression and loss of milestones, sequencing of MECP2 is performed to verify whether Rett syndrome is the cause, by detecting any of the known mutations. Multiplex ligation-dependent probe amplification is also performed to detect major deletions.²⁵

All available treatments for Rett syndrome are symptomatic; intensive early intervention is practiced.³¹ Multidisciplinary management – medical, psychiatric, and physical – is introduced almost immediately after diagnosis. Following diagnosis, patients are prescribed anti-seizure, sleep, and anxiety medications.³¹ Electroencephalography can be performed to identify seizure type. Neuromuscular blockage drugs can be prescribed to help with gait and stereotypic hand movements.²⁵

Handguards or splints to the elbows can be prescribed by an occupational therapist to improve hand movement.²⁵ Physical therapy can improve mobility; hydrotherapy and hippotherapy have been successful in helping to maintain mobility and muscle support.^32,33 Nutritional management is implemented to control caloric intake and maintain the vitamin D level.³¹ Some patients experience constipation and urinary retention, putting them at risk of nephrolithiasis.

Once the signs and symptoms of Rett syndrome progress, and milestones regress to a certain point, patients need constant, full-time care for the rest of their lives.³⁴ As symptomatic interventions have greatly improved patient outcomes and it has been shown that about 70% can reach adulthood with a potential lifespan of about 50 years.²⁵

Although there is no cure for Rett syndrome and treatments are symptomatic, ongoing studies – both clinical and preclinical – offer promise that treatments will be developed that work at molecular and genetic levels.
 

 

Clinical trials

Advances in understanding of Rett syndrome have led to many therapies in clinical trials, several of which show promise.

Trofinetide. One of the most promising targets for downstream therapy, mentioned earlier, is IGF-1, which was the target of a successful phase 3 clinical trial, LAVENDER (sponsored by Acadia Pharmaceuticals).^35,36 This trial studied trofinetide, a synthetic IGF-1 analog that inhibits neuroinflammation, restores glial function, corrects synaptic deficiencies, and regulates oxidative stress response.^12,37,38 Initial results from phase 2 and phase 3 trials indicate improved scores for treated patients in the Rett syndrome Behaviour Questionnaire (RSBQ) and Clinical Global Impression–Improvement (CGI-I) scores, while also showing improvements in the Communication and Symbolic Behavior Scales Developmental Profile Infant–Toddler Checklist–Social composite score.^36,39

The most common adverse events seen with trofinetide were diarrhea and vomiting.

Acadia Pharmaceuticals has filed for approval of a new drug application for trofinetide with the Food and Drug Administration, for which the company has been granted Fast Track Status and orphan drug designations. Most (95%) subjects in the phase 3 LAVENDER trial elected to continue taking trofinetide in the subsequent open-label Lilac and Lilac-2 extension studies.³⁶ A current open-label phase 2/3 trial is recruiting patients 2 to 5 years of age to evaluate trofinetide.⁴⁰ It is expected that, in the near future, this could be a drug given to Rett patients as an FDA-approved treatment.

Blarcamesine. Another small molecule drug, blarcamesine (also known as ANAVEX2-73), a sigma-1 receptor agonist, produced promising results in phase 2 clinical trials in adult Rett syndrome patients. The drug is in a phase 2/3 clinical trial for pediatric Rett syndrome patients (sponsored by Anavex Life Sciences).^41-43

Phase 2 results indicated statistically significant and clinically meaningful improvement in RSBQ and CGI-I scores with blarcamesine. Improvement was initially observed within 4 weeks after the start of treatment and was sustained throughout the study. The drug was shown to be well tolerated, with minimal adverse effects; no serious adverse events were recorded. These results were observed in adult patients, demonstrating that improvements in Rett syndrome are possible even after regression.

Blarcamesine activates the sigma 1 receptor, which is pivotal to restoring cellular homeostasis and restoring neuroplasticity – deficiencies of which have been linked to autophagy and glutamate toxicity. The drug has also been explored as a potential treatment for other neurological disorders.^44-47 Improvements in blarcamesine-treated patients further correlated with lower levels of glutamate in cerebrospinal fluid, which is a Rett syndrome biomarker, supporting the proposition that behavioral improvements were due to drug intervention.^48,49 The phase 2 trial was modified into a phase 3 trial and additional endpoints were added.^41-43

All patients in the phase 2 adult trial elected to continue in the extension study.

Based on these promising data, Anavex is pursuing an approval pathway for adult patients, while continuing dosage optimization phase 2/3 trials and recruitment for a pediatric trial.^42,43

Is the future about gene therapy?

TSHA-102 (miniMECP2). Taysha Gene Therapies is developing a promising gene therapy, TSHA-102, for Rett syndrome, and is aiming to begin phase 1/2 clinical trials in 2022.⁵⁰ The technology for this therapy relies on the delivery of a fragment of MECP2 (known as miniMECP2), which is regulated by a built-in microRNA regulator (miR-responsive auto-regulatory element, or miRARE) to help ameliorate MECP2 dosage toxicity. (Overexpression of MECP2 is toxic to neurons, which has made traditional [so to speak] gene replacement therapy difficult in Rett syndrome: Levels of MECP2 need to be tightly regulated, and the Taysha microRNA technology regulates levels of miniMECP2, thus reducing toxicity.)

 

The Taysha microRNA technology has yielded promising results in mouse studies for Rett syndrome; results indicate a lengthening of lifespan and delayed onset of gait abnormalities.⁵¹ TSHA-102 is in the preclinical stage but offers promise that it will be the first gene therapy for Rett syndrome to enter clinical trials.

As the field of gene therapy advances, several promising technologies are on the horizon that could potentially have disease-altering impacts on Rett syndrome. These therapies are divided into two broad categories: those at the gene level and those at the transcription and protein level. A few of these approaches are highlighted below.

Gene replacement involves adding a full or partial copy of MECP2 to neuronal cells. This type of therapy presents challenges, from delivery of the new gene to dosage concerns, because MECP2 can be toxic if overexpressed.^52-54 Groundbreaking work was done in mouse models involving truncated MECP2, exhibiting phenotypic rescue and validating the gene-replacement approach.¹⁸ This strategy is being pursued by Neurogene, which has a uinique technology that allows for tuning of the gene’s expression to get the correct protein levels in the patient. Promising data was presented this year at the American Society of Gene and Cell Therapy conference.⁵⁵

Early gene replacement therapy studies also laid the foundation for the minMECP2 and microRNA approach being used by Taysha Gene Therapies (discussed above).⁵¹

“Correcting” DNA mutations. A different approach at the genetic level involves “correcting” mutations in MECP2 at the DNA level. This is possible because, in a large subset of Rett syndrome patients who have the same missense or nonsense mutations, by using CRISPR, a gene editing tool (discussed above) a single base pair can be corrected.^56,57 Previous research, in a Rett syndrome-model of induced pluripotent stem cells, showed that this type of editing is possible – and effective.⁵² An approach with particular promise involves use of a class of CRISPR proteins known as base editors that are able to specifically alter a single base of DNA.⁵⁷ The technique has the potential to address many of the mutations seen in Rett syndrome; research on this type of technology is being pursued by Beam Therapeutics, and has the potential to impact Rett syndrome.⁵⁸

Another promising “correction” approach is exonic editing, in which a much larger section of DNA – potentially, exons 3 and 4, which, taken together, comprise 97% of known MECP2 mutations seen in Rett syndrome – are replaced.⁵⁹

In both CRISPR and exonic editing therapeutic approaches, endogenous levels of MECP2 expression would be maintained. Of note, both approaches are being pursued for use in treating other genetic disorders, which provides a boost in scaling-up work on addressing safety and efficacy concerns that accompany gene-editing approaches.⁵⁸ One advantage to the DNA correction approach is that is has the potential to be a “one-and-done” treatment.

“Correcting” RNA. Beyond directly editing DNA, several therapeutic approaches are exploring the ability to edit RNA or to provide the protein directly to cells as enzyme replacement therapy. Such an RNA correction strategy leverages a technology that takes advantage of cells’ natural RNA editor, known as adenosine deaminase acting on RNA (ADAR), which corrects errors in cells’ RNA by providing specific guides to the cell. ADAR can be targeted to fix mutations in the MECP2 RNA transcript, resulting in a “corrected” MECP2 protein.^60,61 This technology has delivered promising proof-of-concept evidence in cells and in murine models, and is in the therapeutic pipeline at VICO Therapeutics.⁶²

 

Shape Therapeutics has also leveraged ADAR to “correct” mutated RNA; Rett syndrome is among the top priorities in the company’s pipeline.

Worth noting is that there are several advantages to the “correction” approach:

• Leveraging internal repair mechanisms minimizes the immune response.
• The flexibility of correction means that it can be used to address many of the mutations that cause Rett syndrome.⁶³

Enzyme replacement therapy, in which the MECP2 protein produced by MECP2 would be directly replaced, is being explored in Rett syndrome patients. This technology has been used successfully in Pompe disease; however, Rett syndrome presents its own challenge because MECP2 needs to be delivered to the brain and neuronal cells.⁶⁴

Where does this work stand? The technologies described in this section are in preclinical stages of study. Nonetheless, it is expected that several will enter human clinical trials during the next 5 years.
 

Conclusion

A diagnosis of Rett syndrome is a life-altering event for patients and their family. But there is more hope than ever for effective therapies and, eventually, a cure.

Multiple late-stage clinical trials in progress are demonstrating promising results from therapeutic products, with minimal adverse events. Remarkably, these interventions have delivered improvements to adult patients after regression has stabilized. With rapid progress being made in the field of gene therapy, several technologies for which are focused on Rett syndrome, a hopeful picture is emerging: that therapeutic intervention will be possible before regression, thus effectively treating and, potentially, even curing Rett syndrome.

The landscape is broadening. Add to this hope for approved therapies is the fact that Rett syndrome isn’t the only target being pursued with such strategies; in fact, researchers in the larger field of neurodevelopmental disorder study are working together to find common solutions to shared challenges – from how therapies are designed and delivered to how toxicity is minimized. Much of what is being explored in the Rett syndrome field is also under investigation in other neurodevelopmental syndromes, including Angelman, Prader-Willi, chromosome 15q11.2-13.1 duplication (dup15q), and Fragile X syndrome. This kind of parallel investigation benefits all parties and optimizes a treatment platform so that it can be applied across more than a single disorder.

Like many monogenic disorders, Rett syndrome is entering an exciting stage – at which the words “treatment” and “cure” can be spoken with intent and vision, not just wide-eyed optimism. These words portend real promise for patients who carry the weight of a diagnosis of Rett syndrome, and for their families.
 

Ms. Ambrose is a student in the master’s of science in human genetics and genomic data analytics program, Keck Graduate Institute, Claremont, Calif. Dr. Bailus is an assistant professor of genetics, Keck Graduate Institute. The authors report no conflict of interest related to this article.

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61. Sinnamon JR et al. Site-directed RNA repair of endogenous Mecp2 RNA in neurons. Proc Natl Acad Sci U S A. 2017 Oct 31;114(44):E9395-E9402. doi: 10.1073/pnas.1715320114.

62. Pipeline. VICO Therapeutics [Internet]. Updated Nov 5, 2021. Accessed Feb 23, 2022. https://vicotx.com/pipeline.

63. Therapeutics platform. Shape Therapeutics [Internet]. Updated Feb 20, 2021. Accessed Feb 23, 2022.

https://live-shapetx.pantheonsite.io/therapeutics-platform.

64. Koeberl DD et al. Glycogen storage disease types I and II: Treatment updates. J Inherit Metab Dis. 2007 Apr;30(2):159-64. doi: 10.1007/s10545-007-0519-9.

The dream of curing genetic disorders has been a persistent but elusive goal, even before the human genome was mapped. Once mapping of the human genome was complete in 2001, an entirely new avenue of potential treatments and cures for genetic diseases and disorders was opened.^1,2

The disorders best suited for targeted gene therapy are monogenic; however, tools and delivery methods for editing the human genome were limited and difficult to apply, until the advent of the CRISPR system in 2012.³ CRISPR (an acronym of clustered regularly interspaced short palindromic repeats) has changed the way in which gene therapy strategies are pursued, with dozens of companies leveraging a variety of platforms to create potentially life-changing therapies for devastating rare diseases and disorders.

One of the rare monogenic disorders that is embracing multiple gene therapy approaches is Rett syndrome, a rare, debilitating neurodevelopmental disorder. In this review, we explore the molecular cause of Rett syndrome, disease presentation, current treatments, ongoing clinical trials, and therapies that are on the horizon.
 

Underlying molecular cause

Rett syndrome is caused by mutations in, or the absence of, the MECP2 gene, which produces methyl-CpG binding protein 2 (MECP2). The syndrome was first described clinically in 1954 by the Austrian physician Andreas Rett; it would take until 1982 before the disorder was officially named, eponymously, in a seminal paper by Hagberg.⁴ After Hagberg’s characterization, Rett syndrome became the predominant global clinical diagnosis identified among cognitively impaired females, with an incidence of 1 in every 10,000 to 15,000.⁴

In 1999, mutations in, and deletions of, MECP2 were identified as the cause of Rett syndrome.^4,5 MECP2 is located on the X chromosome, in the Xq28 region, making Rett syndrome an X-linked dominant disorder.⁶ Rett syndrome is seen predominantly in females who are mosaic for mutant or deleted MECP2. Random X chromosome inactivation results in some cells expressing the mutant MECP2 allele and other cells expressing the normal functioning MECP2 allele; the percentage of cells expressing the normal allele correlates with the degree of syndrome severity.^7-9

The incidence of Rett syndrome is much lower in males, in whom the syndrome was originally thought to be lethal; many observed male cases are either mosaic or occur in XXY males.^10,11

Approximately 95% of cases of Rett syndrome are due to de novo mutations in MECP2, with a handful of specific mutations and large deletions accounting for more than 85% of cases.¹² The fact that Rett syndrome is monogenic and most cases are caused by, in total, only a handful of mutations or deletions makes the syndrome a promising candidate for gene therapy.

At the molecular level, it has been observed that the MECP2 mutations of Rett syndrome lead to loss of gene function, thus disrupting the ability of the MECP2 nuclear protein to regulate global gene transcription through its binding to methylated DNA sites.¹² A large percentage of these missense and nonsense mutations lead to a truncated or nonfunctional protein.¹²

One of the ways in which MECP2 regulates transcription is as a component of heterochromatin condensates and by separation of heterochromatin and euchromatin.^13-15 It has been observed that the cells of Rett syndrome patients have an altered chromatin state, potentially contributing to transcriptional dysregulation.^16,17 Several mutations observed in Rett syndrome patients occur in crucial domains for heterochromatin condensate formation, which helps explain this cellular phenotype.¹³ Introduction of a engineered “mini” MECP2 in a murine model of Rett syndrome has resulted in partial rescue of heterochromatin condensate formation and transcriptional regulation – fostering the hypothesis that correcting those genetic changes could lead to a potential therapy.¹⁸

Beyond the role of MECP2 in heterochromatin condensate formation, the gene interacts with more than 40 proteins that have diverse roles in cellular function, epigenetic modulation, and neuronal development. This volume of interactions contributes to MECP2 being a global gene regulatory protein that has far-reaching effects on transcriptional regulation across the genome.^19-22

Epigenetic dysregulation has been associated with neurodevelopmental and neuropsychiatric disorders.²³ Both insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor are transcriptional targets of MECP2, and are involved in neuronal differentiation, synaptic function, and neurite outgrowth.¹² This helps explain the neurodevelopmental phenotypes observed in MECP2-mutated patients.

Notably, although Rett syndrome patients experience neurodevelopmental phenotypes at the cellular level, neuronal death is not readily observed. That observation provides hope that an interventional therapy after onset of symptoms might still be of benefit.
 

 

Presentation

Early neurotypical development. A hallmark of Rett syndrome is neurotypical physical and mental development until 6 to 24 months of age.

Stagnation is the first stage of the syndrome, involving a small but rapid decline in habitual milestones, such motor and language skills.¹² Subtle signs, such as microcephaly and hypotonia, can also arise at this time but might be missed.²⁴

Rapid regression follows stagnation. Speech and motor delays and impaired gait and breathing occur;^12,25 purposeful hand skills are lost, replaced by repetitive hand-wringing movements that are a hallmark of the syndrome.^12,24 Seizures are observed; they become more common during the next stage.¹²

Plateau. Language advances can be observed, but further deficits are seen in motor skills and hand coordination.¹²

Late motor deterioration stage. Late physical deficits develop, leading to lifelong impairments. The physical deficits observed are the result of severe muscle weakness, usually resulting in wheelchair dependency.¹²

Plateau. Patients then reach a second plateau. Regression stops; deficient physical and cognitive states stabilize and are maintained.²⁵

At all stages of Rett syndrome, the following are observed:

• Gastrointestinal problems.
• Sleep disturbances.
• Abnormal cardiorespiratory coupling.
• Greater-than-expected mortality.¹²

Final regression. The patient is fully dependent for the rest of their lifespan, partially due to seizure activity.^26,27
 

A life-changing diagnosis

A diagnosis of Rett syndrome is life-changing for a patient’s family; access to supportive groups of other patients and their families is extremely beneficial. Two helpful organizations – the Rett Syndrome Research Trust²⁸ and International Rett Syndrome Foundation,²⁹ – offer patient support and community and fund research.

Because X chromosome inactivation is random in Rett syndrome, the individual patient can present with a wide variety of phenotypic combinations – making the patient, and their needs, unique.¹² During stages of regression, patients often experience emotional dysregulation and anxiety, which is attributable to their increasing physical difficulties.³⁰ They often exhibit combinations of uncontrolled movements, including repetitive rocking, scratching, and self-injurious behavior.³⁰ For most, regression subsides after the first 5 years of alternating development and regression; after that, their ultimate symptoms persist for life.²⁵

As patients mature, they need to be monitored for proper nutrition and scoliosis.²⁵ As adults, they are at risk of pneumonia, respiratory distress, status epilepticus, osteopenia, and lack of adequate food or water because of impaired ability to feed.²⁵

The lifespan of Rett syndrome patients has increased, thanks to improvements in health care, advances in technology, and early genetic testing, which allows for earlier diagnosis, intervention, and management of symptoms.
 

Current treatments

When a female patient presents with regression and loss of milestones, sequencing of MECP2 is performed to verify whether Rett syndrome is the cause, by detecting any of the known mutations. Multiplex ligation-dependent probe amplification is also performed to detect major deletions.²⁵

All available treatments for Rett syndrome are symptomatic; intensive early intervention is practiced.³¹ Multidisciplinary management – medical, psychiatric, and physical – is introduced almost immediately after diagnosis. Following diagnosis, patients are prescribed anti-seizure, sleep, and anxiety medications.³¹ Electroencephalography can be performed to identify seizure type. Neuromuscular blockage drugs can be prescribed to help with gait and stereotypic hand movements.²⁵

Handguards or splints to the elbows can be prescribed by an occupational therapist to improve hand movement.²⁵ Physical therapy can improve mobility; hydrotherapy and hippotherapy have been successful in helping to maintain mobility and muscle support.^32,33 Nutritional management is implemented to control caloric intake and maintain the vitamin D level.³¹ Some patients experience constipation and urinary retention, putting them at risk of nephrolithiasis.

Once the signs and symptoms of Rett syndrome progress, and milestones regress to a certain point, patients need constant, full-time care for the rest of their lives.³⁴ As symptomatic interventions have greatly improved patient outcomes and it has been shown that about 70% can reach adulthood with a potential lifespan of about 50 years.²⁵

Although there is no cure for Rett syndrome and treatments are symptomatic, ongoing studies – both clinical and preclinical – offer promise that treatments will be developed that work at molecular and genetic levels.
 

 

Clinical trials

Advances in understanding of Rett syndrome have led to many therapies in clinical trials, several of which show promise.

Trofinetide. One of the most promising targets for downstream therapy, mentioned earlier, is IGF-1, which was the target of a successful phase 3 clinical trial, LAVENDER (sponsored by Acadia Pharmaceuticals).^35,36 This trial studied trofinetide, a synthetic IGF-1 analog that inhibits neuroinflammation, restores glial function, corrects synaptic deficiencies, and regulates oxidative stress response.^12,37,38 Initial results from phase 2 and phase 3 trials indicate improved scores for treated patients in the Rett syndrome Behaviour Questionnaire (RSBQ) and Clinical Global Impression–Improvement (CGI-I) scores, while also showing improvements in the Communication and Symbolic Behavior Scales Developmental Profile Infant–Toddler Checklist–Social composite score.^36,39

The most common adverse events seen with trofinetide were diarrhea and vomiting.

Acadia Pharmaceuticals has filed for approval of a new drug application for trofinetide with the Food and Drug Administration, for which the company has been granted Fast Track Status and orphan drug designations. Most (95%) subjects in the phase 3 LAVENDER trial elected to continue taking trofinetide in the subsequent open-label Lilac and Lilac-2 extension studies.³⁶ A current open-label phase 2/3 trial is recruiting patients 2 to 5 years of age to evaluate trofinetide.⁴⁰ It is expected that, in the near future, this could be a drug given to Rett patients as an FDA-approved treatment.

Blarcamesine. Another small molecule drug, blarcamesine (also known as ANAVEX2-73), a sigma-1 receptor agonist, produced promising results in phase 2 clinical trials in adult Rett syndrome patients. The drug is in a phase 2/3 clinical trial for pediatric Rett syndrome patients (sponsored by Anavex Life Sciences).^41-43

Phase 2 results indicated statistically significant and clinically meaningful improvement in RSBQ and CGI-I scores with blarcamesine. Improvement was initially observed within 4 weeks after the start of treatment and was sustained throughout the study. The drug was shown to be well tolerated, with minimal adverse effects; no serious adverse events were recorded. These results were observed in adult patients, demonstrating that improvements in Rett syndrome are possible even after regression.

Blarcamesine activates the sigma 1 receptor, which is pivotal to restoring cellular homeostasis and restoring neuroplasticity – deficiencies of which have been linked to autophagy and glutamate toxicity. The drug has also been explored as a potential treatment for other neurological disorders.^44-47 Improvements in blarcamesine-treated patients further correlated with lower levels of glutamate in cerebrospinal fluid, which is a Rett syndrome biomarker, supporting the proposition that behavioral improvements were due to drug intervention.^48,49 The phase 2 trial was modified into a phase 3 trial and additional endpoints were added.^41-43

All patients in the phase 2 adult trial elected to continue in the extension study.

Based on these promising data, Anavex is pursuing an approval pathway for adult patients, while continuing dosage optimization phase 2/3 trials and recruitment for a pediatric trial.^42,43

Is the future about gene therapy?

TSHA-102 (miniMECP2). Taysha Gene Therapies is developing a promising gene therapy, TSHA-102, for Rett syndrome, and is aiming to begin phase 1/2 clinical trials in 2022.⁵⁰ The technology for this therapy relies on the delivery of a fragment of MECP2 (known as miniMECP2), which is regulated by a built-in microRNA regulator (miR-responsive auto-regulatory element, or miRARE) to help ameliorate MECP2 dosage toxicity. (Overexpression of MECP2 is toxic to neurons, which has made traditional [so to speak] gene replacement therapy difficult in Rett syndrome: Levels of MECP2 need to be tightly regulated, and the Taysha microRNA technology regulates levels of miniMECP2, thus reducing toxicity.)

 

The Taysha microRNA technology has yielded promising results in mouse studies for Rett syndrome; results indicate a lengthening of lifespan and delayed onset of gait abnormalities.⁵¹ TSHA-102 is in the preclinical stage but offers promise that it will be the first gene therapy for Rett syndrome to enter clinical trials.

As the field of gene therapy advances, several promising technologies are on the horizon that could potentially have disease-altering impacts on Rett syndrome. These therapies are divided into two broad categories: those at the gene level and those at the transcription and protein level. A few of these approaches are highlighted below.

Gene replacement involves adding a full or partial copy of MECP2 to neuronal cells. This type of therapy presents challenges, from delivery of the new gene to dosage concerns, because MECP2 can be toxic if overexpressed.^52-54 Groundbreaking work was done in mouse models involving truncated MECP2, exhibiting phenotypic rescue and validating the gene-replacement approach.¹⁸ This strategy is being pursued by Neurogene, which has a uinique technology that allows for tuning of the gene’s expression to get the correct protein levels in the patient. Promising data was presented this year at the American Society of Gene and Cell Therapy conference.⁵⁵

Early gene replacement therapy studies also laid the foundation for the minMECP2 and microRNA approach being used by Taysha Gene Therapies (discussed above).⁵¹

“Correcting” DNA mutations. A different approach at the genetic level involves “correcting” mutations in MECP2 at the DNA level. This is possible because, in a large subset of Rett syndrome patients who have the same missense or nonsense mutations, by using CRISPR, a gene editing tool (discussed above) a single base pair can be corrected.^56,57 Previous research, in a Rett syndrome-model of induced pluripotent stem cells, showed that this type of editing is possible – and effective.⁵² An approach with particular promise involves use of a class of CRISPR proteins known as base editors that are able to specifically alter a single base of DNA.⁵⁷ The technique has the potential to address many of the mutations seen in Rett syndrome; research on this type of technology is being pursued by Beam Therapeutics, and has the potential to impact Rett syndrome.⁵⁸

Another promising “correction” approach is exonic editing, in which a much larger section of DNA – potentially, exons 3 and 4, which, taken together, comprise 97% of known MECP2 mutations seen in Rett syndrome – are replaced.⁵⁹

In both CRISPR and exonic editing therapeutic approaches, endogenous levels of MECP2 expression would be maintained. Of note, both approaches are being pursued for use in treating other genetic disorders, which provides a boost in scaling-up work on addressing safety and efficacy concerns that accompany gene-editing approaches.⁵⁸ One advantage to the DNA correction approach is that is has the potential to be a “one-and-done” treatment.

“Correcting” RNA. Beyond directly editing DNA, several therapeutic approaches are exploring the ability to edit RNA or to provide the protein directly to cells as enzyme replacement therapy. Such an RNA correction strategy leverages a technology that takes advantage of cells’ natural RNA editor, known as adenosine deaminase acting on RNA (ADAR), which corrects errors in cells’ RNA by providing specific guides to the cell. ADAR can be targeted to fix mutations in the MECP2 RNA transcript, resulting in a “corrected” MECP2 protein.^60,61 This technology has delivered promising proof-of-concept evidence in cells and in murine models, and is in the therapeutic pipeline at VICO Therapeutics.⁶²

 

Shape Therapeutics has also leveraged ADAR to “correct” mutated RNA; Rett syndrome is among the top priorities in the company’s pipeline.

Worth noting is that there are several advantages to the “correction” approach:

• Leveraging internal repair mechanisms minimizes the immune response.
• The flexibility of correction means that it can be used to address many of the mutations that cause Rett syndrome.⁶³

Enzyme replacement therapy, in which the MECP2 protein produced by MECP2 would be directly replaced, is being explored in Rett syndrome patients. This technology has been used successfully in Pompe disease; however, Rett syndrome presents its own challenge because MECP2 needs to be delivered to the brain and neuronal cells.⁶⁴

Where does this work stand? The technologies described in this section are in preclinical stages of study. Nonetheless, it is expected that several will enter human clinical trials during the next 5 years.
 

Conclusion

A diagnosis of Rett syndrome is a life-altering event for patients and their family. But there is more hope than ever for effective therapies and, eventually, a cure.

Multiple late-stage clinical trials in progress are demonstrating promising results from therapeutic products, with minimal adverse events. Remarkably, these interventions have delivered improvements to adult patients after regression has stabilized. With rapid progress being made in the field of gene therapy, several technologies for which are focused on Rett syndrome, a hopeful picture is emerging: that therapeutic intervention will be possible before regression, thus effectively treating and, potentially, even curing Rett syndrome.

The landscape is broadening. Add to this hope for approved therapies is the fact that Rett syndrome isn’t the only target being pursued with such strategies; in fact, researchers in the larger field of neurodevelopmental disorder study are working together to find common solutions to shared challenges – from how therapies are designed and delivered to how toxicity is minimized. Much of what is being explored in the Rett syndrome field is also under investigation in other neurodevelopmental syndromes, including Angelman, Prader-Willi, chromosome 15q11.2-13.1 duplication (dup15q), and Fragile X syndrome. This kind of parallel investigation benefits all parties and optimizes a treatment platform so that it can be applied across more than a single disorder.

Like many monogenic disorders, Rett syndrome is entering an exciting stage – at which the words “treatment” and “cure” can be spoken with intent and vision, not just wide-eyed optimism. These words portend real promise for patients who carry the weight of a diagnosis of Rett syndrome, and for their families.
 

Ms. Ambrose is a student in the master’s of science in human genetics and genomic data analytics program, Keck Graduate Institute, Claremont, Calif. Dr. Bailus is an assistant professor of genetics, Keck Graduate Institute. The authors report no conflict of interest related to this article.

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56. Anzalone AV et al. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol. 2020 Jul;38(7):824-44. doi: 10.1038/s41587-020-0561-9.

57. Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A●T to G●C in genomic DNA without DNA cleavage. Nature. 2017 Nov 23;551(7681):464-71. doi: 10.1038/nature24644.

58. Coenraads M. How RSRT is driving the search for a Rett cure. Rett Syndrome Research Trust [Internet]. Dec 7, 2021. Accessed Feb 23, 2022. https://rettnews.org/articles/how-rsrt-is-driving-the-search-for-a-rett-cure.

59. Cutting-edge technologies to repair the underlying mutations that cause Rett. Rett Syndrome Research Trust [Internet]. Updated Nov 3, 2021. Accessed Feb 23, 2022. https://reverserett.org/research/cures/gene-editing.

60. Sinnamon JR et al. In vivo repair of a protein underlying a neurological disorder by programmable RNA editing. Cell Rep. 2020 Jul 14;32(2):107878. doi: 10.1016/j.celrep.2020.107878.

61. Sinnamon JR et al. Site-directed RNA repair of endogenous Mecp2 RNA in neurons. Proc Natl Acad Sci U S A. 2017 Oct 31;114(44):E9395-E9402. doi: 10.1073/pnas.1715320114.

62. Pipeline. VICO Therapeutics [Internet]. Updated Nov 5, 2021. Accessed Feb 23, 2022. https://vicotx.com/pipeline.

63. Therapeutics platform. Shape Therapeutics [Internet]. Updated Feb 20, 2021. Accessed Feb 23, 2022.

https://live-shapetx.pantheonsite.io/therapeutics-platform.

64. Koeberl DD et al. Glycogen storage disease types I and II: Treatment updates. J Inherit Metab Dis. 2007 Apr;30(2):159-64. doi: 10.1007/s10545-007-0519-9.

The dream of curing genetic disorders has been a persistent but elusive goal, even before the human genome was mapped. Once mapping of the human genome was complete in 2001, an entirely new avenue of potential treatments and cures for genetic diseases and disorders was opened.^1,2

The disorders best suited for targeted gene therapy are monogenic; however, tools and delivery methods for editing the human genome were limited and difficult to apply, until the advent of the CRISPR system in 2012.³ CRISPR (an acronym of clustered regularly interspaced short palindromic repeats) has changed the way in which gene therapy strategies are pursued, with dozens of companies leveraging a variety of platforms to create potentially life-changing therapies for devastating rare diseases and disorders.

One of the rare monogenic disorders that is embracing multiple gene therapy approaches is Rett syndrome, a rare, debilitating neurodevelopmental disorder. In this review, we explore the molecular cause of Rett syndrome, disease presentation, current treatments, ongoing clinical trials, and therapies that are on the horizon.
 

Underlying molecular cause

Rett syndrome is caused by mutations in, or the absence of, the MECP2 gene, which produces methyl-CpG binding protein 2 (MECP2). The syndrome was first described clinically in 1954 by the Austrian physician Andreas Rett; it would take until 1982 before the disorder was officially named, eponymously, in a seminal paper by Hagberg.⁴ After Hagberg’s characterization, Rett syndrome became the predominant global clinical diagnosis identified among cognitively impaired females, with an incidence of 1 in every 10,000 to 15,000.⁴

In 1999, mutations in, and deletions of, MECP2 were identified as the cause of Rett syndrome.^4,5 MECP2 is located on the X chromosome, in the Xq28 region, making Rett syndrome an X-linked dominant disorder.⁶ Rett syndrome is seen predominantly in females who are mosaic for mutant or deleted MECP2. Random X chromosome inactivation results in some cells expressing the mutant MECP2 allele and other cells expressing the normal functioning MECP2 allele; the percentage of cells expressing the normal allele correlates with the degree of syndrome severity.^7-9

The incidence of Rett syndrome is much lower in males, in whom the syndrome was originally thought to be lethal; many observed male cases are either mosaic or occur in XXY males.^10,11

Approximately 95% of cases of Rett syndrome are due to de novo mutations in MECP2, with a handful of specific mutations and large deletions accounting for more than 85% of cases.¹² The fact that Rett syndrome is monogenic and most cases are caused by, in total, only a handful of mutations or deletions makes the syndrome a promising candidate for gene therapy.

At the molecular level, it has been observed that the MECP2 mutations of Rett syndrome lead to loss of gene function, thus disrupting the ability of the MECP2 nuclear protein to regulate global gene transcription through its binding to methylated DNA sites.¹² A large percentage of these missense and nonsense mutations lead to a truncated or nonfunctional protein.¹²

One of the ways in which MECP2 regulates transcription is as a component of heterochromatin condensates and by separation of heterochromatin and euchromatin.^13-15 It has been observed that the cells of Rett syndrome patients have an altered chromatin state, potentially contributing to transcriptional dysregulation.^16,17 Several mutations observed in Rett syndrome patients occur in crucial domains for heterochromatin condensate formation, which helps explain this cellular phenotype.¹³ Introduction of a engineered “mini” MECP2 in a murine model of Rett syndrome has resulted in partial rescue of heterochromatin condensate formation and transcriptional regulation – fostering the hypothesis that correcting those genetic changes could lead to a potential therapy.¹⁸

Beyond the role of MECP2 in heterochromatin condensate formation, the gene interacts with more than 40 proteins that have diverse roles in cellular function, epigenetic modulation, and neuronal development. This volume of interactions contributes to MECP2 being a global gene regulatory protein that has far-reaching effects on transcriptional regulation across the genome.^19-22

Epigenetic dysregulation has been associated with neurodevelopmental and neuropsychiatric disorders.²³ Both insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor are transcriptional targets of MECP2, and are involved in neuronal differentiation, synaptic function, and neurite outgrowth.¹² This helps explain the neurodevelopmental phenotypes observed in MECP2-mutated patients.

Notably, although Rett syndrome patients experience neurodevelopmental phenotypes at the cellular level, neuronal death is not readily observed. That observation provides hope that an interventional therapy after onset of symptoms might still be of benefit.
 

 

Presentation

Early neurotypical development. A hallmark of Rett syndrome is neurotypical physical and mental development until 6 to 24 months of age.

Stagnation is the first stage of the syndrome, involving a small but rapid decline in habitual milestones, such motor and language skills.¹² Subtle signs, such as microcephaly and hypotonia, can also arise at this time but might be missed.²⁴

Rapid regression follows stagnation. Speech and motor delays and impaired gait and breathing occur;^12,25 purposeful hand skills are lost, replaced by repetitive hand-wringing movements that are a hallmark of the syndrome.^12,24 Seizures are observed; they become more common during the next stage.¹²

Plateau. Language advances can be observed, but further deficits are seen in motor skills and hand coordination.¹²

Late motor deterioration stage. Late physical deficits develop, leading to lifelong impairments. The physical deficits observed are the result of severe muscle weakness, usually resulting in wheelchair dependency.¹²

Plateau. Patients then reach a second plateau. Regression stops; deficient physical and cognitive states stabilize and are maintained.²⁵

At all stages of Rett syndrome, the following are observed:

• Gastrointestinal problems.
• Sleep disturbances.
• Abnormal cardiorespiratory coupling.
• Greater-than-expected mortality.¹²

Final regression. The patient is fully dependent for the rest of their lifespan, partially due to seizure activity.^26,27
 

A life-changing diagnosis

A diagnosis of Rett syndrome is life-changing for a patient’s family; access to supportive groups of other patients and their families is extremely beneficial. Two helpful organizations – the Rett Syndrome Research Trust²⁸ and International Rett Syndrome Foundation,²⁹ – offer patient support and community and fund research.

Because X chromosome inactivation is random in Rett syndrome, the individual patient can present with a wide variety of phenotypic combinations – making the patient, and their needs, unique.¹² During stages of regression, patients often experience emotional dysregulation and anxiety, which is attributable to their increasing physical difficulties.³⁰ They often exhibit combinations of uncontrolled movements, including repetitive rocking, scratching, and self-injurious behavior.³⁰ For most, regression subsides after the first 5 years of alternating development and regression; after that, their ultimate symptoms persist for life.²⁵

As patients mature, they need to be monitored for proper nutrition and scoliosis.²⁵ As adults, they are at risk of pneumonia, respiratory distress, status epilepticus, osteopenia, and lack of adequate food or water because of impaired ability to feed.²⁵

The lifespan of Rett syndrome patients has increased, thanks to improvements in health care, advances in technology, and early genetic testing, which allows for earlier diagnosis, intervention, and management of symptoms.
 

Current treatments

When a female patient presents with regression and loss of milestones, sequencing of MECP2 is performed to verify whether Rett syndrome is the cause, by detecting any of the known mutations. Multiplex ligation-dependent probe amplification is also performed to detect major deletions.²⁵

All available treatments for Rett syndrome are symptomatic; intensive early intervention is practiced.³¹ Multidisciplinary management – medical, psychiatric, and physical – is introduced almost immediately after diagnosis. Following diagnosis, patients are prescribed anti-seizure, sleep, and anxiety medications.³¹ Electroencephalography can be performed to identify seizure type. Neuromuscular blockage drugs can be prescribed to help with gait and stereotypic hand movements.²⁵

Handguards or splints to the elbows can be prescribed by an occupational therapist to improve hand movement.²⁵ Physical therapy can improve mobility; hydrotherapy and hippotherapy have been successful in helping to maintain mobility and muscle support.^32,33 Nutritional management is implemented to control caloric intake and maintain the vitamin D level.³¹ Some patients experience constipation and urinary retention, putting them at risk of nephrolithiasis.

Once the signs and symptoms of Rett syndrome progress, and milestones regress to a certain point, patients need constant, full-time care for the rest of their lives.³⁴ As symptomatic interventions have greatly improved patient outcomes and it has been shown that about 70% can reach adulthood with a potential lifespan of about 50 years.²⁵

Although there is no cure for Rett syndrome and treatments are symptomatic, ongoing studies – both clinical and preclinical – offer promise that treatments will be developed that work at molecular and genetic levels.
 

 

Clinical trials

Advances in understanding of Rett syndrome have led to many therapies in clinical trials, several of which show promise.

Trofinetide. One of the most promising targets for downstream therapy, mentioned earlier, is IGF-1, which was the target of a successful phase 3 clinical trial, LAVENDER (sponsored by Acadia Pharmaceuticals).^35,36 This trial studied trofinetide, a synthetic IGF-1 analog that inhibits neuroinflammation, restores glial function, corrects synaptic deficiencies, and regulates oxidative stress response.^12,37,38 Initial results from phase 2 and phase 3 trials indicate improved scores for treated patients in the Rett syndrome Behaviour Questionnaire (RSBQ) and Clinical Global Impression–Improvement (CGI-I) scores, while also showing improvements in the Communication and Symbolic Behavior Scales Developmental Profile Infant–Toddler Checklist–Social composite score.^36,39

The most common adverse events seen with trofinetide were diarrhea and vomiting.

Acadia Pharmaceuticals has filed for approval of a new drug application for trofinetide with the Food and Drug Administration, for which the company has been granted Fast Track Status and orphan drug designations. Most (95%) subjects in the phase 3 LAVENDER trial elected to continue taking trofinetide in the subsequent open-label Lilac and Lilac-2 extension studies.³⁶ A current open-label phase 2/3 trial is recruiting patients 2 to 5 years of age to evaluate trofinetide.⁴⁰ It is expected that, in the near future, this could be a drug given to Rett patients as an FDA-approved treatment.

Blarcamesine. Another small molecule drug, blarcamesine (also known as ANAVEX2-73), a sigma-1 receptor agonist, produced promising results in phase 2 clinical trials in adult Rett syndrome patients. The drug is in a phase 2/3 clinical trial for pediatric Rett syndrome patients (sponsored by Anavex Life Sciences).^41-43

Phase 2 results indicated statistically significant and clinically meaningful improvement in RSBQ and CGI-I scores with blarcamesine. Improvement was initially observed within 4 weeks after the start of treatment and was sustained throughout the study. The drug was shown to be well tolerated, with minimal adverse effects; no serious adverse events were recorded. These results were observed in adult patients, demonstrating that improvements in Rett syndrome are possible even after regression.

Blarcamesine activates the sigma 1 receptor, which is pivotal to restoring cellular homeostasis and restoring neuroplasticity – deficiencies of which have been linked to autophagy and glutamate toxicity. The drug has also been explored as a potential treatment for other neurological disorders.^44-47 Improvements in blarcamesine-treated patients further correlated with lower levels of glutamate in cerebrospinal fluid, which is a Rett syndrome biomarker, supporting the proposition that behavioral improvements were due to drug intervention.^48,49 The phase 2 trial was modified into a phase 3 trial and additional endpoints were added.^41-43

All patients in the phase 2 adult trial elected to continue in the extension study.

Based on these promising data, Anavex is pursuing an approval pathway for adult patients, while continuing dosage optimization phase 2/3 trials and recruitment for a pediatric trial.^42,43

Is the future about gene therapy?

TSHA-102 (miniMECP2). Taysha Gene Therapies is developing a promising gene therapy, TSHA-102, for Rett syndrome, and is aiming to begin phase 1/2 clinical trials in 2022.⁵⁰ The technology for this therapy relies on the delivery of a fragment of MECP2 (known as miniMECP2), which is regulated by a built-in microRNA regulator (miR-responsive auto-regulatory element, or miRARE) to help ameliorate MECP2 dosage toxicity. (Overexpression of MECP2 is toxic to neurons, which has made traditional [so to speak] gene replacement therapy difficult in Rett syndrome: Levels of MECP2 need to be tightly regulated, and the Taysha microRNA technology regulates levels of miniMECP2, thus reducing toxicity.)

 

The Taysha microRNA technology has yielded promising results in mouse studies for Rett syndrome; results indicate a lengthening of lifespan and delayed onset of gait abnormalities.⁵¹ TSHA-102 is in the preclinical stage but offers promise that it will be the first gene therapy for Rett syndrome to enter clinical trials.

As the field of gene therapy advances, several promising technologies are on the horizon that could potentially have disease-altering impacts on Rett syndrome. These therapies are divided into two broad categories: those at the gene level and those at the transcription and protein level. A few of these approaches are highlighted below.

Gene replacement involves adding a full or partial copy of MECP2 to neuronal cells. This type of therapy presents challenges, from delivery of the new gene to dosage concerns, because MECP2 can be toxic if overexpressed.^52-54 Groundbreaking work was done in mouse models involving truncated MECP2, exhibiting phenotypic rescue and validating the gene-replacement approach.¹⁸ This strategy is being pursued by Neurogene, which has a uinique technology that allows for tuning of the gene’s expression to get the correct protein levels in the patient. Promising data was presented this year at the American Society of Gene and Cell Therapy conference.⁵⁵

Early gene replacement therapy studies also laid the foundation for the minMECP2 and microRNA approach being used by Taysha Gene Therapies (discussed above).⁵¹

“Correcting” DNA mutations. A different approach at the genetic level involves “correcting” mutations in MECP2 at the DNA level. This is possible because, in a large subset of Rett syndrome patients who have the same missense or nonsense mutations, by using CRISPR, a gene editing tool (discussed above) a single base pair can be corrected.^56,57 Previous research, in a Rett syndrome-model of induced pluripotent stem cells, showed that this type of editing is possible – and effective.⁵² An approach with particular promise involves use of a class of CRISPR proteins known as base editors that are able to specifically alter a single base of DNA.⁵⁷ The technique has the potential to address many of the mutations seen in Rett syndrome; research on this type of technology is being pursued by Beam Therapeutics, and has the potential to impact Rett syndrome.⁵⁸

Another promising “correction” approach is exonic editing, in which a much larger section of DNA – potentially, exons 3 and 4, which, taken together, comprise 97% of known MECP2 mutations seen in Rett syndrome – are replaced.⁵⁹

In both CRISPR and exonic editing therapeutic approaches, endogenous levels of MECP2 expression would be maintained. Of note, both approaches are being pursued for use in treating other genetic disorders, which provides a boost in scaling-up work on addressing safety and efficacy concerns that accompany gene-editing approaches.⁵⁸ One advantage to the DNA correction approach is that is has the potential to be a “one-and-done” treatment.

“Correcting” RNA. Beyond directly editing DNA, several therapeutic approaches are exploring the ability to edit RNA or to provide the protein directly to cells as enzyme replacement therapy. Such an RNA correction strategy leverages a technology that takes advantage of cells’ natural RNA editor, known as adenosine deaminase acting on RNA (ADAR), which corrects errors in cells’ RNA by providing specific guides to the cell. ADAR can be targeted to fix mutations in the MECP2 RNA transcript, resulting in a “corrected” MECP2 protein.^60,61 This technology has delivered promising proof-of-concept evidence in cells and in murine models, and is in the therapeutic pipeline at VICO Therapeutics.⁶²

 

Shape Therapeutics has also leveraged ADAR to “correct” mutated RNA; Rett syndrome is among the top priorities in the company’s pipeline.

Worth noting is that there are several advantages to the “correction” approach:

• Leveraging internal repair mechanisms minimizes the immune response.
• The flexibility of correction means that it can be used to address many of the mutations that cause Rett syndrome.⁶³

Enzyme replacement therapy, in which the MECP2 protein produced by MECP2 would be directly replaced, is being explored in Rett syndrome patients. This technology has been used successfully in Pompe disease; however, Rett syndrome presents its own challenge because MECP2 needs to be delivered to the brain and neuronal cells.⁶⁴

Where does this work stand? The technologies described in this section are in preclinical stages of study. Nonetheless, it is expected that several will enter human clinical trials during the next 5 years.
 

Conclusion

A diagnosis of Rett syndrome is a life-altering event for patients and their family. But there is more hope than ever for effective therapies and, eventually, a cure.

Multiple late-stage clinical trials in progress are demonstrating promising results from therapeutic products, with minimal adverse events. Remarkably, these interventions have delivered improvements to adult patients after regression has stabilized. With rapid progress being made in the field of gene therapy, several technologies for which are focused on Rett syndrome, a hopeful picture is emerging: that therapeutic intervention will be possible before regression, thus effectively treating and, potentially, even curing Rett syndrome.

The landscape is broadening. Add to this hope for approved therapies is the fact that Rett syndrome isn’t the only target being pursued with such strategies; in fact, researchers in the larger field of neurodevelopmental disorder study are working together to find common solutions to shared challenges – from how therapies are designed and delivered to how toxicity is minimized. Much of what is being explored in the Rett syndrome field is also under investigation in other neurodevelopmental syndromes, including Angelman, Prader-Willi, chromosome 15q11.2-13.1 duplication (dup15q), and Fragile X syndrome. This kind of parallel investigation benefits all parties and optimizes a treatment platform so that it can be applied across more than a single disorder.

Like many monogenic disorders, Rett syndrome is entering an exciting stage – at which the words “treatment” and “cure” can be spoken with intent and vision, not just wide-eyed optimism. These words portend real promise for patients who carry the weight of a diagnosis of Rett syndrome, and for their families.
 

Ms. Ambrose is a student in the master’s of science in human genetics and genomic data analytics program, Keck Graduate Institute, Claremont, Calif. Dr. Bailus is an assistant professor of genetics, Keck Graduate Institute. The authors report no conflict of interest related to this article.

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