A Blueprint for Translational Precision Medicine in Autism Spectrum Disorder and Related Neurogenetic Syndromes

Abstract

Objectives:

Despite growing knowledge of the underlying neurobiology of autism spectrum disorder (ASD) and related neurogenetic syndromes, treatment discovery has remained elusive. In this review, we provide a blueprint for translational precision medicine in ASD and related neurogenetic syndromes.

Methods:

The discovery of trofinetide for Rett syndrome (RTT) is described, and the role of nonmammalian, mammalian, and stem cell model systems in the identification of molecular targets and drug screening is discussed. We then provide a framework for translating preclinical findings to human clinical trials, including the role of biomarkers in selecting molecular targets and evaluating target engagement, and discuss how to leverage these findings for future ASD drug development.

Results:

Multiple preclinical model systems for ASD have been developed, each with tradeoffs with regard to suitability for high-throughput small molecule screening, conservation across species, and behavioral face validity. Future clinical trials should incorporate biomarkers and intermediate phenotypes to demonstrate target engagement. Factors that contributed to the approval of trofinetide for RTT included replicated findings in mouse models, a well-studied natural history of the syndrome, development of RTT-specific outcome measures, and strong engagement of the RTT family community.

Conclusions:

The translation of our growing understanding of the neurobiology of ASD to human drug discovery will require a precision medicine approach, including the use of multiple model systems for molecular target selection, evaluation of target engagement, and clinical trial design strategies that address heterogeneity, power, and the placebo response.

Introduction

Autism spectrum disorder (ASD) is an etiologically and clinically heterogeneous developmental disorder that is behaviorally characterized by deficits in social communication, along with restricted interests, repetitive behaviors, and/or sensory sensitivities. Many neurogenetic syndromes, for example, Fragile X syndrome (FraX), have long been associated with increased rates of ASD, and more recent evidence has increased support for the importance of genetics in ASD. Despite a growing knowledge of the underlying neurobiology of ASD and related neurogenetic syndromes, no medications have consistently demonstrated efficacy for the treatment of the core symptoms of ASD. In this review, we provide a blueprint for translational precision medicine in ASD and related neurogenetic syndromes. We begin by describing trofinetide in individuals with Rett syndrome (RTT) as an example of translational precision medicine. We then describe the role of nonmammalian, mammalian, and stem cell model systems in the identification of molecular targets and drug screening. We present a framework for translating these findings to human clinical trials, including the role of neuroimaging-based and other human biomarkers of ASD in selecting molecular targets and evaluating target engagement, and discuss how to leverage these findings considering clinical trial design, the orphan drug approval process, and finally, accessibility of newly approved therapies and their dissemination to the community.

Trofinetide in Rett syndrome as a case example of precision medicine development

Trofinetide, a synthetic glycine–proline–glutamate (GPE) analog of insulin-like growth factor-1 (IGF-1), was approved by the U.S. Food and Drug Administration (FDA) for the treatment of RTT in adults and children aged 2 years and older in March 2023. RTT is a severe neurodevelopmental disorder (NDD) that predominantly affects females and is characterized by the partial or complete loss of purposeful hand skills and spoken language, gait abnormalities, and stereotypic hand movements such as hand wringing/squeezing and clapping/tapping (Neul et al., 2010). The symptoms of RTT evolve across the lifespan and typically progress through four stages (Neul et al., 2010). Developmental progress slows or stalls between ages 6 and 18 months (early onset phase), which is accompanied by a dramatic deceleration in brain growth. This is followed by a regression in purposeful hand movements and expressive language between 1 and 4 years of age (rapid destructive phase). Regression generally slows around 2 years of age, whereas seizures and other medical comorbidities, such as gastrointestinal problems and breathing disturbances, become the primary concern (plateau phase). In adolescence and adulthood, individuals can experience stiffness or loss of muscle tone, as well as breathing abnormalities (late motor deterioration phase).

RTT affects approximately 1 in 10,000–15,000 live births, and more than 95% of cases are caused by loss-of-function mutations of the methyl-CpG-binding protein 2 (MECP2) gene. While the mechanism by which MECP2 mutations cause RTT has not been fully elucidated, the gene product, MeCP2 protein, is known to be a transcriptional regulator and chromatin remodeler that is essential for normal brain development as well as the maintenance of neuronal function in adulthood (McGraw et al., 2011). Over 200 different MECP2 mutations have been associated with RTT (Cuddapah et al., 2014), and loss-of-function or partial loss-of-function MECP2 mutations have also been associated with ASD, intellectual disability (ID), or specific learning disorders in both males and females (Pascual-Alonso et al., 2021).

Considering the multisystem nature of RTT, clinical management has traditionally centered on managing individual issues symptomatically, often including polypharmacy. More recently, researchers have sought agents with the potential to target multiple core symptoms of RTT by addressing the loss of MeCP2 function.

Clinical observations of the developmental regression and deceleration in brain growth in RTT led to the hypothesis that neuroprotective agents might be helpful in ameliorating its core symptoms. IGF-1 is a naturally occurring, hepatically synthesized growth factor that crosses the blood–brain barrier. IGF-1 has long been recognized as having neuroprotective properties based on its involvement in neuronal regeneration and survival, as well as its procognitive and antiaging effects (Lichtenwalner et al., 2001; Markowska et al., 1998; Popken et al., 2004; Trejo et al., 2007). A limitation of its use pharmacologically, however, is its short half-life. GPE is a neuroactive degradation product of IGF-1. Similar to IGF-1, GPE also has a very short half-life, which led to the synthesis of trofinetide (initially known as NNZ-2566), with improved stability and bioavailability, as a candidate drug for neuroprotection. Trofinetide is available as an oral solution that is typically administered twice per day.

Mouse models of RTT have been utilized to investigate the behavioral and mechanistic effects of IGF-1, GPE, and trofinetide. One study found reduced levels of IGF-1 in Mecp2 null mice, and treatment of these animals with recombinant IGF-1 resulted in improved lifespan, locomotor activity, heart rate, respiration patterns, and social and anxiety behaviors (Castro et al., 2014). In another study, GPE was shown to extend the lifespan, improve locomotor function, and ameliorate abnormal breathing patterns, as well as restore dendritic spine density and stabilize cortical plasticity in Mecp2 mutant mice (Tropea et al., 2009). Trofinetide itself has primarily been assessed in murine models of brain injury, such as ischemia and traumatic brain injury, and has been shown to attenuate apoptotic cell death (Bickerdike et al., 2009) and to reduce serum levels of injury-mediated inflammatory cytokines (Wei et al., 2009).

The development, validation, and regulatory acceptance of high-quality outcome measures were necessary to translate these promising mouse findings into clinical trials in humans with RTT. The Clinical Global Impression Improvement subscale (CGI-I) and the Rett Syndrome Behavior Questionnaire (RSBQ) were the two coprimary outcome measures in the phase III trial that were submitted to the FDA, ultimately resulting in approval of trofinetide for the treatment of RTT. The CGI-I is a clinician-rated scale that has been used in multiple populations as an FDA-accepted measure of global clinical change to establish the real-world impact of a novel treatment (Guy, 1976). In contrast, the RSBQ was developed as a caregiver-rated scale to assess the severity of neurobehavioral problems specifically associated with RTT (Mount et al., 2002). It was originally written as a diagnostic tool to differentiate females with RTT from those with other etiologies of ID. The RSBQ was first used as a clinical trial assessment measure in a placebo-controlled crossover trial of recombinant human IGF-1 (mecasermin) for RTT (O'Leary et al., 2018) and was later accepted by the FDA as a primary outcome measure for clinical trials in RTT (Percy, 2023). The RSBQ consists of 45 items, with 38 grouped into 8 subscales: general mood; breathing problems; hand behaviors; repetitive face movements; body rocking and expressionless face; nighttime behaviors; fear/anxiety; and walking/standing. The remaining seven items contribute to the RSBQ total score but are uncategorized. Although the CGI-I is widely used in clinical trials as an index of clinically meaningful improvement in the real world, it lacks specificity for RTT. Therefore, key anchors for CGI-I ratings specific to RTT were developed in preparation for phase II trials. These anchors and accompanying sample RTT vignettes were based on symptom duration, onset, durability of change, and sign/symptom change across symptom domains (Neul et al., 2015).

Phase 1 and 2 clinical trials of trofinetide were sponsored by Neuren Pharmaceuticals. The pharmacokinetics of trofinetide were investigated in a phase 1 clinical trial of 61 healthy subjects, demonstrating linear pharmacokinetics and a half-life of 1.4 hours, indicating the need for twice or three times per day dosing (Oosterholt et al., 2017). This was followed by two phase 2 clinical trials, RETT-01 (Glaze et al., 2017) and RETT-002 (Glaze et al., 2019). RETT-01 was an exploratory, multicenter, double-blind, placebo-controlled, dose-escalation study of the safety and tolerability of trofinetide in 56 females with RTT (15.9–44.2 years) for up to 28 days (Glaze et al., 2017). Both doses of trofinetide (35 mg/kg twice daily and 70 mg/kg twice daily) were safe and well tolerated. The most common adverse effects were diarrhea, irritability, and somnolence (Glaze et al., 2017), and no serious adverse effects were related to trofinetide. RETT-02 was a multicenter, double-blind, placebo-controlled, parallel-group study that included 82 children and adolescents with RTT (5.1–15.9 years) who were randomized in a 1:1:1:1 ratio to placebo, 50, 100, or 200 mg/kg of trofinetide twice per day for 42 days (Glaze et al., 2019). All dose levels were well tolerated and safe, with only one participant withdrawing from the study due to gastrointestinal side effects (Glaze et al., 2019). The 200 mg/kg dose of trofinetide was associated with statistically significant improvements on the RSBQ total score, RTT-Clinician Domain Specific Concerns-Visual Analog Scale, and the CGI-I compared with placebo (Glaze et al., 2019).

Acadia Pharmaceuticals then acquired the rights to trofinetide in North America and executed the phase 3 clinical trial program, including facilitating participant logistics such as transportation and accommodation during trial visits, as well as drug shipment to families’ homes to minimize participant burden (Kennedy et al., 2024). The program consisted of LAVENDAR, a randomized, double-blind, placebo-controlled trial of trofinetide in 184 females with RTT (5–20 years; Neul et al., 2023), and two open-label extensions for up to 32 months (Percy et al., 2024a). LAVENDAR randomized females with RTT to twice-daily trofinetide 200 mg (n = 93) or placebo (n = 94) for 12 weeks. There was a statistically significant trofinetide-placebo difference on both coprimary outcome measures, with a least squares mean (LSM) change from baseline to week 12 on the RSBQ of −4.9 versus −1.7 (p = 0.0175, Cohen’s d 0.37) and LSM CGI-I at week 12 of 3.5 versus 3.8 (p = 0.003, Cohen’s d 0.47). Diarrhea (80.6%) and vomiting (26.9%) were the most common treatment-emergent adverse events. There was also significant treatment benefit for trofinetide compared with placebo on communication scales (Neul et al., 2024). Symptoms of RTT continued to improve in the open-label extensions (Percy et al., 2024b).

To assess the role of trofinetide in younger patients, DAFFODIL enrolled 2–4-year-old girls with RTT in a phase 2, open-label trial evaluating safety and efficacy over two treatment phases (Percy et al., 2023). The first phase was 12 weeks long and evaluated dosing, safety, and efficacy, whereas the second phase assessed longer-term safety and efficacy for approximately 21 months. The results of the first phase were consistent with those from the LAVENDAR study, supporting a New Drug Application and the approval of trofinetide in pediatric patients as young as 2 years.

Beyond the study participants, investigators, and industry sponsors, the International Rett Syndrome Foundation (IRSF) played a pivotal role in the ultimate success of trofinetide in RTT. Critical contributions from the IRSF included partially funding the Rett Syndrome Natural History study, which not only served as a foundation for developing specific clinical trial outcome measures but also developed trusted relationships between investigators and families. The IRSF also helped identify clinical sites for trials; partially funded early clinical trials of trofinetide; and facilitated participant recruitment by providing education to families on clinical trial design, including eligibility criteria and the purpose of a placebo arm (Kennedy et al., 2024). Finally, the IRSF disseminated information about the trial results to families and continues to play an ongoing, instrumental role in elevating the voices of affected families as research for a cure continues.

Nonhuman Model Systems

Studies in preclinical model systems, including nonmammalian, mammalian, and human stem cells, are critical for identifying pharmacological pathways and molecular targets relevant to ASD and related neurogenetic syndromes. Here, we describe the unique features of each system that make them well-suited for advancing translational drug discovery.

Nonmammalian models

Preclinical studies may include less complex animal systems, including Drosophila melanogaster (fruit flies), Caenorhabditis elegans (nematodes), Danio rerio (zebrafish), and Xenopus laevis (African clawed frog), all of which have several key advantages for genetic and pharmacological studies relevant to ASD and NDDs. First, they are highly tractable and can be studied at a lower cost compared with rodents. Second, given their relative simplicity, it is possible to conduct analyses at the whole-organism level during early developmental stages (McCammon and Sive, 2015). For example, zebrafish have fully transparent embryos and larvae, which makes it possible for researchers to visualize the effect of a genetic manipulation or drug in an intact, developing nervous system in real time (Ijaz and Hoffman, 2016). Third, these systems have large progenies (e.g., zebrafish can produce ∼100–300 embryos in a single clutch), which makes them amenable to high-throughput screens at a scale that would not be practical in rodents (McCammon and Sive, 2015). Fourth, these systems are relatively easy to manipulate genetically using techniques such as RNA interference (RNAi) and Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated protein 9 (CRISPR)-Cas9. Therefore, it is possible to target hundreds of ASD/NDD-associated genes in parallel at a relatively low cost.

To some extent, invertebrates (Drosophila, C. elegans) and less complex vertebrates (zebrafish, Xenopus) represent an optimal intermediate between human-induced pluripotent stem cell (hiPSC) and rodent models (discussed below), in that they approach the throughput of an in vitro system while having an intact nervous system, allowing for assessments of behavior, whole-brain structure, and circuitry (McCammon and Sive, 2015). However, as with any animal system, there are limitations to their translatability to humans. As McCammon and Sive (2015) observe, there is a tradeoff between tractability and conservation in these systems (McCammon and Sive, 2015). Invertebrates are the most amenable to large-scale, rapid genetic, and drug/toxicity screens yet are less evolutionarily conserved compared with humans (McCammon and Sive, 2015). Drosophila and C. elegans have homologs of 75% and ∼50% of human disease genes, respectively (Culetto and Sattelle, 2000; McCammon and Sive, 2015; Reiter et al., 2001). Zebrafish and Xenopus share ∼80% of human disease genes, yet assays involving these systems tend to be more medium-throughput and involve a longer timeframe (McCammon and Sive, 2015; Nenni et al., 2019).

Despite the evolutionary distance to humans, there is a remarkable degree of conservation of neurobiological mechanisms in these systems, which makes them translationally relevant to humans. While zebrafish and Xenopus lack a prefrontal cortex, they have the same major subdivisions of a vertebrate brain as humans—forebrain, midbrain, and hindbrain (Exner and Willsey, 2021; Kozol et al., 2016). In addition, zebrafish have the same neurotransmitter systems and major neural cell types as humans, and studies have demonstrated the conservation of developmental marker gene expression (Guo, 2009), pharmacological pathways (Guo, 2009; Prober et al., 2006; Rihel et al., 2010), and neural circuits underlying basic behaviors (Burgess and Granato, 2007a, 2007b; Lovett-Barron et al., 2017; Schoonheim et al., 2010) in zebrafish and humans. For example, a screen of >1000 psychoactive drugs in zebrafish larvae revealed conserved effects of compounds on sleep behaviors (Rihel et al., 2010). Another study used live brain imaging in zebrafish larvae to identify a group of neuromodulatory neurons controlling alertness; next, they showed that direct activation of related neuronal populations in mice controlled a similar behavior, demonstrating the deep conservation of neuromodulatory circuits in zebrafish and mice (Lovett-Barron et al., 2017). Therefore, given the advantages of these systems for high-throughput studies, combined with a reasonable degree of conservation, they are well-suited for first-pass screens aimed at identifying genetic mechanisms and drug candidates with translational relevance to humans.

Recent studies have leveraged scalable animal systems to analyze the function of multiple ASD/NDD-associated genes in parallel. For example, by using RNAi to knock down 14 genes in the 16p11.2del interval in Drosophila, one study identified interactions among gene pairs within the 16p11.2del interval and between these genes and other core neurodevelopmental genes in controlling cell proliferation phenotypes in the developing fly eye (Iyer et al., 2018). This study provides evidence for combinatorial effects of genes within ASD/NDD-associated copy number variants (CNVs) on basic neurodevelopmental phenotypes (Iyer et al., 2018). Another study used a real-time computer vision system called the Multi-Worm Tracker (Swierczek et al., 2011) to quantify behaviors in C. elegans mutants of almost 100 ASD-associated genes (McDiarmid et al., 2020). Interestingly, they identified unique effects of disrupting each gene on locomotion, tactile responses, and habituation learning and were able to cluster genes by similarities in their phenotypic profiles (McDiarmid et al., 2020). In addition, Willsey et al. (2021) found that disrupting 10 large-effect ASD genes in the developing Xenopus brain using CRISPR/Cas9 affects telencephalon size and the ratio of neural progenitor cells to neurons. Importantly, this study revealed neurogenesis as a novel point of convergence across multiple ASD genes (Willsey et al., 2021). Our group studied 10 large-effect ASD genes in zebrafish mutants and found that disrupting these genes impacts forebrain size, consistent with altered neurogenesis, as well as baseline activity in regions involved in sensory-motor control, such as the thalamus (Weinschutz Mendes et al., 2023). Interestingly, by using large-scale, automated assays of sleep-wake (Prober et al., 2006; Rihel et al., 2010) and visual-startle behaviors (Scott et al., 2016; Weinschutz Mendes et al., 2023) in zebrafish larvae, we identified subgroups of ASD genes with shared behavioral effects (Weinschutz Mendes et al., 2023). Therefore, parallel in vivo screens of multiple ASD/NDD-associated genes have the potential to identify common biological pathways and stratify genes by functional effects, which may provide a path toward targeted drug discovery.

Scalable animal systems have also been used for pharmacological screens targeting ASD/NDD gene-associated phenotypes. For example, Jamadagni et al. (2021) screened 3850 compounds in C. elegans mutants of CHD7, the gene that is disrupted in CHARGE syndrome, to identify suppressors of locomotor deficits (Jamadagni et al., 2021). The top hits were then validated in zebrafish chd7 mutants, leading to the identification of ephedrine, an adrenergic agonist, as a lead compound that ameliorates behavioral abnormalities and GABAergic neuron deficits in both C. elegans and zebrafish (Jamadagni et al., 2021). In addition, our group used pharmacobehavioral profiling in zebrafish mutants of the ASD- and epilepsy-associated gene, CNTNAP2, to predict and test suppressors of a behavioral phenotype and identified estrogenic compounds as novel suppressors of nighttime hyperactivity in zebrafish cntnap2 mutants (Hoffman et al., 2016). Intriguingly, in a screen of 133 U.S. FDA-approved oncology drugs in Xenopus, Willsey et al. (2021) found that compounds affecting estrogen signaling suppressed neural proliferation phenotypes associated with disruption of the ASD gene, DYRK1A. Rescue of proliferation phenotypes by estrogens was validated for select ASD genes in human neural progenitor cells in vitro (Willsey et al., 2021). Collectively, these studies suggest that estrogens may act as modulators of dysregulated pathways downstream of ASD-associated genes. Together, they highlight the advantages of scalable in vivo systems for conducting ASD/NDD gene-based drug screens and identifying novel pharmacological pathways for further evaluation.

Finally, scalable in vivo systems have already been used successfully for drug discovery in other disorders. For example, zebrafish have played an important role in identifying targetable pathways in melanoma (Patton et al., 2021). Zebrafish knock-in models carrying a common BRAF mutation found in melanoma were used to screen for genetic interactors and uncovered SETDB1 as a novel target (Ceol et al., 2011; Patton et al., 2021). Therefore, it is anticipated that with the identification of >250 ASD/NDD-associated genes, scalable in vivo systems will play an important role in stratifying genes with shared biological effects and identifying drug candidates targeting these subgroups for further investigation in rodent and hiPSC models.

Mammalian models

Small vertebrate mammals, primarily mice (Mus musculus) and rats (Rattus norvegicus) but also voles (Microtus ochrogaster) and nonhuman primates (including Rhesus macaque and Macaca fascicularis), have become an integral part of the scientific mission to uncover the underlying biology of neural development. Conservation between humans and rodents at the genomic level of the exome and regulome (noncoding regions of the genome with conserved regulatory information) is substantial (Goodman et al., 2023). Further, the cell types, physiology, and neuronal circuitry of rodents are similar to that of humans and follow roughly similar developmental milestones (Rakic, 2009; Rakic et al., 2009; Silbereis et al., 2016). Mouse and human cortices develop in a series of cell migrations from the inner to the outer layer, eventually forming six cortical layers that show similar regionally specific expression patterns and supporting cell types that form and maintain a blood–brain barrier to protect the neuronal compartment (Sun and Hevner, 2014).

At an individual level, divergent pathways of neurodevelopment can result from an accumulation of additive factors that increase the likelihood of ASD/NDDs and cause a shift in the neurodevelopmental trajectory (Iverson et al., 2023). In the “ice cube” model of the multifactorial nature of ASD/NDDs etiologies, the likelihood of diagnosis is conceptualized as a half-full glass of water: each factor leading to an increase in the likelihood of ASD/NDDs contributes more volume, as happens when adding ice to the glass, and when the glass overflows, the diagnostic threshold for ASD/NDDs is surpassed. Stressors with small effect sizes (such as common variant single nucleotide polymorphisms (Grove et al., 2019) or exposure to environmental pollutants (Modabbernia et al., 2017)) act like ice chips, and only a substantial number of these can overflow the diagnostic threshold. Stressors of large-effect size (such as damaging mutations in dosage-dependent genes) cause a larger increase in the water level, and each one dramatically increases the likelihood of crossing the diagnostic threshold. Several hundred dosage-dependent genes and approximately one dozen recurrent ASD/NDD-associated CNVs have reached genome-wide significance and greatly increase the likelihood of an ASD/NDD diagnosis when they are disrupted (Fu et al., 2022; Zhou et al., 2022), as does in utero exposure to the antiseizure medication valproic acid (Christensen et al., 2013). Each of these factors is considered to have a large and independent effect size.

Large-effect size factors are particularly amenable to being studied in animal models for the following reasons: (1) many of the genes and loci associated with the likelihood of ASD/NDD are conserved or syntenic in smaller mammals, and mutating the homologous genes or loci in animals can cause similar effects on the body; (2) the embryonic environment is contained within the amniotic sac and nurtured through the placenta, permitting the study of prenatal exposures to infections or substances in a similar physiological milieu; (3) the lifespan of small rodents is on a shorter time scale, with growth from conception to adulthood occurring in approximately 2–3 months for mice and 3–4 months for rats; (4) the maturational stages of rodents and nonhuman primates roughly correspond to those in humans; and (5) the similarity of their physiology, metabolic needs, and responses to exogenous substances to that of humans allows for preclinical testing of therapeutic agents with the ability to test different treatments in a larger range of ages.

An array of rapidly growing resources is available to facilitate research in small mammals. The complete genomes of multiple inbred strains of mice and rats have been mapped, and portals such as the University of California Santa Cruz (UCSC) genome browser (Perez et al., 2025) allow analysis of base-pair level conservation, predicted regulatory regions, as well as gene isoform and expression levels in specific tissues with precisely defined age. Genetic CRISPR/Cas engineering tools developed over the past decade have increased the ability and speed of researchers to delete, insert, or substitute new genetic material into the germline to generate novel designer rodents and nonhuman primates (Horie and Nishimori, 2022; Niu et al., 2014; Ran et al., 2013; Shao et al., 2014). With these tools, researchers can not only generate one animal line with a single deletion in a particular gene, but they can also develop lines with an array of different mutations seen in the corresponding clinical cohort and assess each for effect(s) on a panoply of behavioral, molecular, electrophysiologic, and cellular phenotypes. The vast majority of the identified ASD/NDD-associated genes have 1:1 orthologues in smaller mammals, and a subset of ASD/NDD-associated CNVs have corresponding syntenic regions (Fu et al., 2022; Zhou et al., 2022). Mouse models are often the initial preclinical approach to evaluate cross-species validity, due to the existing resources and infrastructure that are readily available and the extensive decades of prior basic research exploring the biology of the mouse. Although environmental models of autism risk have also been employed (Haddad et al., 2020; Haida et al., 2019; Malkova et al., 2012; Tartaglione et al., 2019), here we will focus on genetic models because they are more likely to translate to precision medicine approaches.

Rare genetic mutations are frequently modeled in animals, with MECP2 (RTT), SHANK3 (Phelan–McDermid syndrome), FMR1 (FraX), and CHD8 (CHD8 overgrowth and microdeletion syndrome) being among the most frequently modeled single gene NDDs in mice (as reported by gene.sfari.org). While nonhuman primates offer the most similar biology to humans, nonhuman primate models require more resources to house and study, need a longer time to reach maturity than rodent models, and have fewer research tools readily available for use once the model is generated. Despite these obstacles, several genetic syndromes with higher rates of ASD/NDDs have been modeled in nonhuman primates, including mutations in MECP2, SHANK3, and CHD8 (Chen et al., 2001, 2017; Li et al., 2023a; Liu et al., 2014). Animal models have also been generated to model CNVs implicated in ASD/NDDs, including deletion of 22q11.2, both microdeletions and microduplications of 16p11.2, 7q11.23, 17p11.2, 3q29, 15q13.3, and duplication of 15q11-q13. Once generated, animal models can be assessed using a battery of behavioral tests to assess locomotion, repetitive movements, exploratory drive, memory, and social behaviors. Importantly, the goal in mammalian models is not behavioral assessment for its own sake but to assess these in parallel with in vivo and ex vivo measures of electrophysiology, neuroimaging, and immunohistochemistry to detect brain structure and connectivity. These can be complemented by high-resolution mapping of regulatory, transcriptional, and protein information at the level of tissue samples or in single cells/nuclei.

In many animal models, critical elements of human genetic syndromes are recapitulated. Mice and nonhuman primates engineered to mutate a copy of MECP2 exhibit progressive onset of tremors, seizures, decreased social contacts, and motor abnormalities (including the stereotypic hand wringing that is pathognomonic of RTT), as well as characteristic changes in heart rate and breathing (Chen et al., 2001, 2017; Liu et al., 2014; Shahbazian et al., 2002). Mouse models of CHD8 haploinsufficiency demonstrate an increase in brain size and weight, a decrease in gut motility, and a decrease in locomotion and exploratory behaviors (Bernier et al., 2014; Gompers et al., 2017; Katayama et al., 2016; Platt et al., 2017), while nonhuman primates lacking one copy of CHD8 similarly showed an enlarged brain driven by gliogenesis (Li et al., 2023a). Mice generated to model deletion or duplication of the rCNV 16p11.2 are illustrative of the “Goldilocks” effect (Qureshi et al., 2014), in which a phenotype is observed both when there is too much expression and when there is too little expression of a dosage-dependent gene or CNV. In carriers of either del16p11.2 or dup16p11.2, there is an increased likelihood of ASD/NDDs, but obesity and macrocephaly are a characteristic of the deleted state, while the duplicated state is associated with microcephaly and a smaller body habitus (Qureshi et al., 2014; Zufferey et al., 2012).

Researchers have also endeavored to rescue these phenotypes using pharmaceutical compounds, including some currently in clinical use and others that are experimental. del16p11.2 and dup16p11.2 mice showed similar abnormalities in the KCTD13-Cul3-RhoA pathway (Lin et al., 2015; Lin et al., 2015), implying that one biological pathway may be affected by opposing genetic changes and suggesting the possibility of common molecular targets across distinct genetic syndromes. The γ-aminobutyric acid–B receptor agonist, R-baclofen, has been tested in mice with del16p11.2 (Stoppel et al., 2018), as have antagonists or negative allosteric modulators of the metabotropic glutamate receptor 5 (mGluR5) in mouse models of FraX (Michalon et al., 2012).

As genetic studies of ASD/NDD cohorts have continued, more disruptive mutations in additional genes have been identified. This has highlighted a compelling new avenue for intervention: therapy to target the gene mutation directly. Researchers have adapted genetic engineering technologies to address this goal in several ways. Injecting an adenovirus containing a Mecp2 “minigene” restores the expression of Mecp2 in RTT model mice, although there have also been some difficulties with administration into the central nervous system (CNS) (Luoni et al., 2020; Sinnett et al., 2017). Mouse models of SCN2A and SCN1A haploinsufficiency have shown improvement in neuronal excitability and seizure rates when treated with CRISPR-mediated activation targeting the promoter of the intact gene (Colasante et al., 2020; Wang et al., 2024). Antisense oligonucleotide (ASO)-mediated therapies target the RNA transcripts to either correct mutant alleles (by correcting splicing, for example) or stabilize mRNA made from the nonmutant allele. Using this method, animal models of SCN1A loss (Dravet syndrome) and UBE3A loss (Angelman syndrome [AS]) have shown rescue of the respective phenotypes in mice (Hsiao et al., 2016; Meng et al., 2015), with the latter therapy currently being assessed for therapeutic use in clinical trials of AS patients.

Stem cells and organoids

Stem cell models make it possible to directly test the causal relationships between genotype and phenotype in ASD/NDDs. These in vitro models typically use hiPSCs to produce neurons (Zhang et al., 2013) and glia (McQuade et al., 2018; Tcw et al., 2017). These techniques can be coupled with CRISPR-based genetic (Deneault et al., 2018; Hazelbaker et al., 2017) and genomic (Ho et al., 2017; Liu et al., 2018) engineering to facilitate isogenic (donor-matched) comparisons. Together, these technologies make it possible to systematically test the effect of introducing or removing patient-specific mutations in the very cell types most impacted by these disorders.

Applications of hiPSC models are increasingly uncovering convergent phenotypes associated with high-risk ASD and NDD mutations (Cederquist et al., 2020; Fernandez Garcia et al., 2024; Paulsen et al., 2022; Pintacuda et al., 2023; Sun et al., 2024; Willsey et al., 2021). Crucially, the nature of these phenotypes can vary based on the size and location of patient-specific mutations. For example, in the case of RTT, patient cells show impairments in mitochondrial function and in neuronal maturation that can vary based on the domain of MECP2 that is mutated (Amir et al., 1999; Tanaka et al., 2014). Likewise, distinct patient-specific loss-of-function and gain-of-function mutations in NRXN1 cause decreased synaptic activity in glutamatergic neurons yet increased synaptic activity in GABAergic neurons, which require distinct strategies to ameliorate (Fernando et al., 2024). Thus, precision medicine may ultimately require stratification by not just gene but also the nature of the mutation in order to achieve individualized treatment strategies, adding nuance to future considerations for precision medicine.

While in vitro models that consider one cell type at a time are particularly well suited to explore the cell-autonomous effects of ASD and NDD mutations (Boggess et al., 2025), the cells in these models are typically immature, lacking the physiological relevance and circuit-like activity observed in more complex three-dimensional organoid models. Many of these weaknesses can be addressed through studies of organoid and assembloid models (reviewed in Kelley and Pașca, 2022), which capture the impact of complex circuit- and activity-dependent phenotypes. Large screens of genetic mutations in these models identified abnormalities in cellular proliferation and migration as shared phenotypes resulting from mutations in ASD and NDD risk genes (Li et al., 2023b; Meng et al., 2023). Frequently, these risk mutations show abnormal circuit connectivity (Kim et al., 2024; Paulsen et al., 2022) that recapitulates axon tract deficits often observed in humans with ASD and NDDs (Shukla et al., 2010; Zikopoulos and Barbas, 2010). Organoid models of MECP2 mutations show aberrant electrical activity, apparently driven by specific abnormalities in GABAergic interneurons (Samarasinghe et al., 2021; Xiang et al., 2020). Altogether, organoid models better capture the impact of patient mutations within the context of entire neural networks, improving our understanding of how cellular phenotypes associated with NDD mutations can disrupt brain function at the tissue-wide level.

Human cell-based models also permit fast and efficient screening of potential therapeutics. For example, a pharmacological screen in engineered MECP2 knockout cerebral organoids found that the compounds, nefiracetam and PHA 543613, rescued aberrant phenotypes (Trujillo et al., 2021). Drugs identified in human neuronal models may be well-suited for translating to efficacy in patients. For example, a recent drug screen in patient-derived cells harboring a DISC1 mutation found that the phosphodiesterase-4 inhibitor, rolipram, was able to rescue phenotypes in vitro. Knock-in mice with the same mutation were subsequently treated with rolipram, which rescued both synaptic and behavioral phenotypes (Kim et al., 2021). Thus, stem cell-based models are critical tools in drug development for ASD and NDDs, perhaps particularly in the case of NDDs resulting from known mutations.

Stem cell-based models may also serve as useful tools for the development and delivery of gene therapies. For example, a recent report describes the use of a novel ASO therapy to correct for pathological alternative splicing in the risk gene, CACNA1C, in patient-derived cells, which was also successful in modulating exon usage in vivo in rat brain (Chen et al., 2024). Another study leveraged the respective strengths of stem cell and mouse models to test gene therapy for a mutation in the AS gene, UBE3A. In this experiment, human hematopoietic stem cells were engineered to express mouse Ube3a and were subsequently grafted into mice with Ube3a mutations; the treated mice showed a rescue of Ube3a expression and associated phenotypes (Adhikari et al., 2021). This research provides a model for how gene therapy may be introduced postnatally for some monogenic diseases, highlighting stem cells as possible vectors for therapeutic delivery in addition to their utility in preclinical research.

Altogether, stem cell-derived systems have transformed the modeling of genes and phenotypes associated with ASD and NDDs. Findings well recapitulate known ASD phenotypes, such as synaptic and proliferation differences, and also facilitate high-throughput drug screens to test for the potential of new compounds to rescue observed phenotypes in human neuronal models. In vitro models offer a powerful complement to animal models, which offer more insights into behavioral and systems-level impacts of risk genes and potential therapeutics. In tandem with animal-based preclinical research, stem cell-based research may allow for the high-throughput study of patient mutations and potential drugs in a human neuronal model, rapidly advancing the goal of precision medicine for patients with ASD and NDDs.

Clinical Trial Readiness and Translation

Neuroimaging and human biomarkers

Neuroimaging enables a noninvasive assessment of brain structural, functional, and neurochemical markers at the level of the individual. This could allow subtype stratification based on neural endophenotypes, which may help us understand why individuals respond differently to treatment and thereby allow us to prospectively assign individuals to a given treatment. Recently, the investigation of “biotypes” using personalized scores based on functional brain imaging metrics has shown promise in stratifying patients with depression and anxiety (Tozzi et al., 2024).

Neuroimaging biomarkers from many different imaging modalities, including electroencephalography (EEG), positron emission tomography (PET), and magnetic resonance imaging (MRI), have been investigated in ASD. Several putative EEG biomarkers for ASD have been put forth, with one candidate EEG biomarker being a neural response to faces, known as the N170 (Kala et al., 2021). This face-sensitive event-related potential is one promising marker in the multi-site Autism Biomarkers Consortium for Clinical Trials (ABC-CT) (McPartland et al., 2020; Webb et al., 2023). EEG offers ease of use, requiring only scalp electrodes, and can easily be used across development, from infants to adults, and has excellent temporal resolution, but it also has limitations, including poor spatial resolution. Many other studies have leveraged MRI, which can be used to noninvasively investigate brain structure, function, and biochemistry (e.g., using spectroscopy). Structural MRI biomarkers have so far not led to major significant advances in ASD, but they have shown some promise in longitudinal studies in children who have a higher likelihood of developing ASD, namely younger siblings of children with ASD (e.g., Hazlett et al., 2017; Shen et al., 2022).

In terms of CNS drug development, the imaging methodology that is arguably most directly relevant is PET, as it enables the assessment of neurochemistry and pharmacology in vivo. PET is valuable across drug development stages, as it allows for the assessment of disease-specific molecular characteristics and investigation of pharmacokinetics and pharmacodynamics of candidate CNS drugs (Donnelly, 2017; Lee and Farde, 2006). PET imaging can be used across preclinical and clinical studies, which means that candidate proteins or mechanisms discovered in preclinical models can be interrogated in vivo in humans. Another central characteristic of PET imaging is that it enables assessing whether a candidate drug is brain penetrant and to what extent it engages the desired target in the living brain (Donnelly, 2017). PET occupancy studies can be conducted to assess target engagement and to quantify target occupancy based on a dose or blood concentration, which helps inform clinical trial design and drug dosing (Hooker and Carson, 2019; Takano et al., 2016). For example, PET neuroimaging has shown that a minimum occupancy of 65% of dopamine (D₂) receptors is needed to achieve a clinical response with antipsychotics in patients with schizophrenia. Further, occupancy rates over 78% were associated with a high risk for adverse motor effects (Tauscher and Kapur, 2001). A more recent study leveraged PET-informed target engagement to assess κ-opioid receptor antagonism as a therapeutic intervention for anhedonia in the context of the National Institute of Mental Health (NIMH) Fast Fail Trials (FAST) Initiative (Krystal et al., 2020, 2018).

PET imaging can also be used to discover novel molecular markers that may be altered in disease. Combined with a suitable radiotracer that binds a target of interest, PET imaging studies can quantify the density of receptors, transporters, enzymes, or protein aggregates. In ASD, PET studies have been conducted to investigate serotonin, dopamine, and gamma-aminobutyric acid (GABA), as well as glutamate (reviewed in Zürcher et al., 2015). Recent examples of novel targets studied in the field of ASD include synaptic density through assessment of synaptic vesicle glycoprotein 2A (Matuskey et al., 2024) and mitochondrial complex I (Kato et al., 2023).

Beyond neuroimaging, blood biomarkers of oxidative stress, inflammation, and neurotransmitters have been extensively studied in ASD but less commonly assessed in the context of treatment (Parellada et al., 2023). Fine-grained analysis of specific behavior may offer opportunities to evaluate treatment response and certainly offers promise as a complementary approach to screening or diagnosis. One notable example is the use of eye-tracking as a measure of social visual engagement to assist in the diagnosis of ASD in very young children (Jones et al., 2023). Lessons learned from previous biomarker studies in ASD in the context of imaging and beyond include the need for significantly larger sample sizes given the predicted relatively small effect sizes for a disorder such as ASD (Parellada et al., 2023).

Selection of targets, evaluation of target engagement, and clinical trial design

The considerable etiological and clinical heterogeneity of NDDs has made the identification of targetable molecular mechanisms challenging. Fortunately, the recent genetic and preclinical advances discussed above have increased the likelihood of translating our knowledge of disrupted gene-protein pathways into personalized molecular therapies. For example, while more than 200 risk genes have been identified, many converge into a relatively limited number of biological pathways (Vorstman et al., 2014). In this section, we propose a blueprint for advancing from preclinical molecular target selection to evaluating target engagement and ultimately to clinical trial design and clinical outcomes.

Preclinical target selection

As discussed above, multiple model systems for ASD and related disorders in various species have been developed, each with advantages and disadvantages with regard to cost, ease of genetic manipulation, suitability for high-throughput screening, and similarity to the human brain and behavior (Veenstra-VanderWeele et al., 2023). Within and across species, there are also multiple techniques for modeling NDDs, generally rooted in reverse translating known risk factors for ASD in humans into model systems, for example, by genetic manipulation or introducing environmental risk factors such as maternal immune activation. One major limitation common to currently available model systems of ASD, with the exception of nonhuman primates, is behavioral face validity (Veenstra-VanderWeele et al., 2023). For instance, while cognitive and social deficits are a core aspect of the FraX clinical phenotype in humans, comparatively small and inconsistent deficits have been observed in these domains in mouse models of FraX (Berry-Kravis et al., 2018). This raises significant limitations in utilizing mouse models to evaluate the efficacy of pharmacological interventions in these areas. To overcome the limitations of any single model system, we recommend that molecular target selection ideally be validated in multiple model systems. Preclinical model systems should also be used to determine at what stage of development a target is most amenable to molecular intervention.

Rapid advances in the neuroscience of ASD have led to a range of possible molecular targets, raising the critical question of how to select and prioritize targets. There has been some debate as to whether investing in molecular targets related to synaptic abnormalities versus cell signaling will prove more fruitful. NDDs have long been conceptualized as disorders of synaptic homeostasis, with many arguing for evidence of an excitation–inhibition imbalance (Rubenstein and Merzenich, 2003). A recent study also demonstrated an association between genes related to glutamate (the major excitatory neurotransmitter) or GABA (the major inhibitory neurotransmitter) signaling, ASD symptom profiles, and cortical thickness (Hollestein et al., 2023). Furthermore, synaptic proteins may be a more actionable set of targets compared with cell signaling or transcriptional regulation pathways that have wider-ranging effects (Veenstra-VanderWeele et al., 2023). To date, clinical trials focused on synaptic homeostasis have not yielded new drugs for NDDs, although a limited set of compounds have been tested. Neither of the phase 2 trials of the arbaclofen (a GABA-B receptor agonist) in FraX or ASD demonstrated significant benefit compared with placebo (Berry-Kravis et al., 2017; Veenstra-Vanderweele et al., 2017), although the results of follow-up studies have not yet been published. Studies of mGluR5 antagonists did not demonstrate efficacy for behavioral outcomes as measured by the Aberrant Behavior Checklist FXS-specific algorithm (ABC-C(FX)) total score compared with placebo in adults or adolescents (Berry-Kravis et al., 2016), nor did a follow-up study focusing on language learning in preschool-aged children with FXS. These trial results, along with emerging evidence supporting the role of metabolic and cell signaling abnormalities in NDDs, may support expanding targets beyond neurotransmitter receptors to include downstream signaling pathways or gene regulation (Kaufmann et al., 2024).

Advances in our knowledge of the genetics of NDDs have increased hope for the possibility of engaging genetic targets. For example, gene therapy studies are underway in AS, a severe NDD characterized by ID, extremely limited expressive language, and epilepsy (Keary & McDougle, 2023). AS is caused by insufficient expression of the maternally inherited UBE3A gene. Gene therapies for AS propose rescuing UBE3A function, either by activating the nonexpressed paternal copy of the gene or by inserting UBE3A into the genome. In a proof-of-concept study, UBE3A protein levels and cognitive deficits were partially restored in a mouse model of AS by utilizing an ASO that blocks the expression of the UBE3A antisense transcript, thereby increasing expression of the paternal copy of UBE3A (Meng et al., 2015). Two pharmaceutical companies are actively developing ASO treatments for AS, with human clinical trials underway (GeneTx Biotherapeutics/Ultragenyx, GTX-102, NCT04259281, and Ionis/Biogen [ION582]) (Keary & McDougle, 2023). Pharmaceutical companies are also exploring adeno-associated virus-mediated gene replacement for AS (Keary & McDougle, 2023). Gene therapy is not without risks, however. Current gaps in knowledge include ensuring tissue/cell specificity and identifying when along the developmental trajectory gene therapy will be safe and effective (Henderson et al., 2024). Controlling the degree of protein expression is another important consideration, as the consequences of overexpression of gene products are often not well understood and may even be deleterious. For example, the hemizygous deletion of the Williams–Beuren critical region results in Williams syndrome, whereas duplication of this region is associated with 7q11.23 duplication syndrome. Finally, immune responses to gene therapy, particularly when exposed to a second dose of a gene therapy vector, are a serious concern. Immune responses to the viral vector may inhibit the efficacy of virus readministration and may increase the risk of serious adverse events (Henderson et al., 2024). Two deaths in a pediatric adeno-associated virus gene therapy trial for X-linked myotubular myopathy were reported, which were likely due to an adverse immune response to the treatment (Wilson & Flotte, 2020). There remains much to be learned about the role and risks of gene therapy in advancing the treatment of genetic syndromes, which will need to be balanced with the severity of the natural history of the condition and viable alternative treatment options.

Evaluating target engagement

The NIMH FAST initiative supported early pharmacodynamic evaluation of target engagement. FAST was launched in 2012 in response to the number of compounds that demonstrated promise in early-stage trials but failed to show benefit in later-stage clinical trials (NIMH, 2022), with concern that they did not show clear evidence that the drug actually had the desired impact on brain function, let alone behavioral outcomes. FAST emphasized early evaluation of whether the drug engaged its target (e.g., interaction with a specific receptor), and if sufficient target engagement was not demonstrated, the drug would not advance to later-stage clinical trials. One of the three FAST contracts awarded in 2013 was for ASD. Under this contract, AZD7325, a drug targeting the GABA-A α2/α3 receptor subtype, was evaluated in young adults with ASD (Grabb et al., 2016). This trial showed evidence of target engagement on EEG as an indication of pharmacodynamic response (Grabb et al., 2016), although it did not show changes in clinical endpoints, which were also explored. Target engagement can also be evaluated through several other neuroimaging modalities, such as PET imaging to confirm receptor occupancy (as discussed above) or fMRI to validate activation of the brain region and/or circuit of interest. Ideally, the evaluation of target engagement in humans would parallel studies in model systems. The development and validation of cost-effective, translational techniques that can be used to evaluate target engagement across species are a priority for further research.

Intermediate phenotypes, such as cognitive, behavioral, or psychophysical measures, may also demonstrate target engagement in clinical trials. For example, several intermediate neurocognitive phenotypes have demonstrated promise as potential targets for restricted, repetitive patterns of behavior, such as cognitive flexibility (D’cruz et al., 2016) and inhibitory control (Faja & Nelson Darling, 2019; Lopez et al., 2005; Schmitt et al., 2018). Intermediate phenotypic endpoints (e.g., cognitive endpoints, social reward, and eye-tracking) in early phase trials may allow for n-of-1 trials or single-dose acute challenges to establish target engagement and plausibility of mechanism. Such intermediate phenotypes could also serve as a bridge between early-phase studies and larger trials, especially because change in the core, complex features of ASD may be difficult to observe within the typical timespan of clinical trials (McCracken et al., 2021a).

Clinical trial design and clinical outcomes

Key clinical trial design considerations in ASD include addressing heterogeneity, power, and the placebo response. The heterogeneous nature of ASD has posed a significant challenge to drug development, leading to positive responses in a subset of clinical trial participants but statistically nonsignificant benefits at the overall group level. This highlights a need to identify the individuals who may benefit from a particular treatment (Beversdorf, 2016). Subtyping individuals with ASD based on biomarkers such as genetics, neurotransmitter levels, or physiological reactivity that predict response to a treatment may address heterogeneity (Beversdorf, 2016). Beversdorf et al. (2023) propose incorporating a biomarker exploration phase (phase 2m) into clinical trials. Phase 2m studies would include a broad set of biomarkers (including both mechanistic and other biomarkers) in a moderately large population of participants to understand which subjects are most likely to respond to the drug, allowing for a precision medicine approach when designing phase 3 clinical trials. Importantly, the authors note that open-label studies should be avoided in phase 2m trials, as this may inadvertently identify biomarkers that predict placebo-related or nonspecific improvement during treatment.

Adequately powered trials have been a major issue in NDDs. A systematic review of clinical trials for cognitive deficits in genetic disorders identified 169 trials, with a median sample size of 25 (Van Der Vaart et al., 2015), falling substantially short of the ∼150 participants needed for an adequately powered phase 2b or 3 study. Inconclusive trials are of limited clinical utility and even hamper efforts to investigate novel compounds. Adequately powering clinical trials can be a particular challenge for rare diseases. Potential solutions include developing clinical trial consortia, utilizing telehealth as appropriate, providing transportation and accommodation to families for trial visits, collaborating with patient/family advocacy groups, and ensuring that clinical trial funding mechanisms support fully powered trials.

Placebo response decreases the statistical power to detect a drug-placebo difference. Unfortunately, controlled trials of children and adolescents with ASD have yielded placebo response rates of 20–50% (King et al., 2013). This high rate suggests that open-label trials are not a useful gauge of effect sizes and may not be helpful to identify outcome measures because a substantial portion of improvement will be due to placebo response. Factors that may account for placebo response in ASD include continued neurodevelopment across childhood and adolescence, which can be experienced as improvement during a trial; the waxing and waning nature of some symptoms in ASD, with most participants enrolling during a time of greater difficulty; the nonspecific effects of care and structure that come with trial participation, which can lead to improved scores on study measures; as well as the classical explanation of heightened expectations of improvement among parents who enroll their children in research (King et al., 2013). In one study, an analysis of baseline factors that predicted response to placebo demonstrated that participants with greater severity of disruptive behaviors, mood and ASD symptoms, and caregiver strain were less likely to respond to placebo (King et al., 2013). In another study, greater placebo response was seen at nonacademic sites (Tobe et al., 2023), suggesting that expertise with autism evaluation and care may mitigate placebo response. Additional recommendations to manage expectancy bias include single-blind lead-in phases, separating the efficacy rater from the clinician assessing for adverse events and incorporating objective outcome measures wherever possible (McCracken et al., 2021b).

The broad nature of the core symptoms of ASD, coupled with the number of associated symptoms and comorbid physical and psychiatric problems, raises the question of which clinical endpoints ought to be prioritized for drug development. Some relevant clinical outcome domains include social communication, restricted and repetitive patterns of behavior, sensory issues, and cognition. The field currently lacks consensus on clinical outcome measurement and a narrow set of well-validated clinical tools (Anagnostou, 2018). For instance, an expert panel evaluated 38 social communication measures for use as outcome measures in clinical trials and identified six measures that were appropriate for use but all with significant limitations (Anagnostou et al., 2015). Common limitations include the inability to apply measures across the lifespan, floor effects, insensitivity to change, vulnerability to placebo effects, lack of validation across multiple languages, and burden on participants/families. Importantly, more work is also needed to incorporate patient and caregiver perspectives in outcome measures. Finally, the development and validation of clinical outcomes are also contingent on the availability of effective treatments to demonstrate sensitivity to change, and we are unlikely to see broadly accepted outcome measures until we have positive clinical trials.

Getting new treatments to market

Role of the FDA in treatment development

The goal of industry-sponsored therapeutic development efforts is, of course, to secure regulatory approval for marketing the therapeutic. In the United States, regulatory review falls to the FDA.

For autism and related conditions, the development of most new therapeutics will follow the usual phase 1/2/3 framework, although phase 1 studies of gene therapy in healthy volunteers cannot be justified (as is the case for most oncology treatments). For ultimate approval, the FDA requires “substantial evidence” of clinical benefit, which typically means positive results from two “adequate and well-controlled” studies (Food and Drug Administration, 2023a). The FDA’s criteria for such studies include appropriate selection and assignment of subjects to treatment and control groups, adequate measures to minimize bias, well-defined and reliable methods of assessing response, and rigorous, prospectively planned analyses.

The FDA has the statutory prerogative to exercise its judgment in determining what constitutes substantial evidence and deciding whether two positive trials are necessary (Food and Drug Administration, 2023b). This flexibility can be extended in various circumstances, including for “orphan” conditions, which are defined as those with fewer than 200,000 cases in the United States. Exceptions may allow for approval with only a single study—for example, when a single study is statistically very robust and is accompanied by strongly supportive biomarker data. So-called “accelerated approval” can be granted on the basis of a “surrogate endpoint”—a biomarker rather than a direct measure of clinical impact—when the validity of the biomarker is understood to be strong and when the condition is serious or potentially fatal (Brown, 2024).

During the course of a therapeutic’s development, sponsors communicate repeatedly with the FDA. Because the timeline for these communications can sometimes be slower than optimal, the FDA has provisions to facilitate speedier communication and review. These expedited pathways are available under the FDA’s “fast track” or “breakthrough therapy” designations. Breakthrough designation indicates that a treatment has potential for being a breakthrough, not that it has yet been proven. The FDA also has created a “START” (Support for Clinical Trials Advancing Rare Disease Therapeutics) pilot program. The breakthrough designation was applied to Roche’s balovaptan program in autism, which was later halted after negative phase 2 results, while the pilot START program applies to Neurogene’s current gene therapy program for RTT (Cavazzoni and Marks, 2024).

Although not directly related to ASD, recently developed treatments for spinal muscular atrophy (SMA) illustrate the FDA’s potential regulatory actions (Xu et al., 2017). Before the development of these treatments, infantile SMA was fatal, characterized by loss of spinal motor neurons, leading to weakness, muscle wasting, ventilator dependency, and death—only supportive treatments were available. Nusinersen, an ASO, was the first approved treatment for SMA. The FDA recognized SMA as an orphan condition and granted fast track designation to the nusinersen program. Its first clinical trial was an open-label study in 28 children with SMA, aged 2–14 years, providing critical data on the pharmacokinetics of the treatment and early evidence of clinical efficacy. Subsequent studies extended the lower age range for subject enrollment and examined open-label effects on electromyographic recordings and blood markers of disease progression. A pivotal phase 3 study enrolled 121 subjects below 7 months of age, randomized 2:1 to active treatment or sham (skin prick only) (Finkel et al., 2017). An interim analysis, conducted when 82 subjects had sufficient data, showed clear evidence of efficacy. The FDA approved nusinersen on the basis of the phase 3 results and supportive data from the open-label studies. The approval cites effects both on clinical milestones such as sitting, walking, need for assisted ventilation, and death, as well as ratings on a novel scale of motor function.

Subsequent to the development of nusinersen, a gene replacement therapy for SMA was developed: onasemnogene abeparvovec-xioi, an adeno-associated virus 9 vector that contains a transgene coding for the protein that is needed in SMA (Hoy, 2019). Approval of this therapeutic was based on two open-label studies only (i.e., without any blinded, controlled studies). In the first study, 21 subjects were enrolled, all receiving the same (high) dose of the treatment. In the second study, of 15 subjects, 3 received a lower dose than in Study 1. The FDA noted clinical benefits in Study 1 and in the higher dose cohort of Study 2, which would not have been expected in the then better-documented natural history of the disease. The lower dose cohort fared much less well than the higher dose cohort, establishing the higher dose as the minimally effective.

As discussed earlier in this article, the selection of clinical outcome assessments is among the most important decisions in trial design. While there remain important questions about the psychometrics of the existing measures for ASD, the landscape of outcome measures is less clear for genetic conditions associated with severe intellectual and other disabilities. Many of the commonly used measures of child development may show floor effects in these populations or otherwise be unsuitable. Recognizing this gap, the FDA awarded a grant to support the continued development of the Observer-Reported Communication Ability scale, a measure of early communication skills that can be used across modalities of communication (e.g., speech, sign, or alternative/augmented communication device) (Food and Drug Administration, 2024). It is both rare and welcome to see the FDA taking this role in treatment development.

Access and implementation

Funding of treatment development and treatment pricing

The cost of developing a novel medical therapeutic is estimated to range between hundreds of millions of dollars to more than $4 billion (Schlander et al., 2021). These estimates account for all the costs after basic research has identified a biological target, from the identification and synthesis of a molecule that is chemically optimal, to safety and efficacy testing in animal models, to in vitro toxicology, to upscaling of chemical synthesis, through clinical phases 1, 2, and 3. The cost estimates also account for expenditures on failed programs, which far outnumber the programs that successfully garner FDA approval. Some development programs seek to “re-purpose” drugs that have already been studied for other conditions (Pedini et al., 2023). This strategy avoids the costs of work already performed for the original development program (i.e., if the previous program reached clinical testing, then new costs for all the preclinical work would be minimized).

The overwhelming majority of therapeutic development programs are funded by industry, although there exist a handful of academic and charitable efforts to develop personalized, gene-targeted treatments. The pioneering academic effort to develop an ASO for a girl with Batten’s disease resulted in a treatment known as Milasen (the patient was named Mila), but the putative utility of the ASO was limited to the patient’s specific genetic variant in the CLN7 gene (Wilton-Clark et al., 2024). (The efficacy of the ASO for Mila was never established unequivocally. Her seizure frequency appeared to decrease after treatment, but she passed away about 2 years after initiating treatment.)

Since no drug has yet been approved for treating the core symptoms of autism, the market pricing of any such treatment remains speculative—perhaps such treatments will be priced similarly to other novel, small-molecule treatments for conditions such as arthritis or diabetes. Treatments for orphan conditions are commonly priced much higher, on the premise that the manufacturer must recoup its costs (and make a profit) on a much smaller number of patients and doses. For example, onasemnogene, the gene therapy for SMA that has dramatic benefits that are hoped will last a lifetime, is estimated to cost over $2 million for a single administration (Fields, 2025). Trofinetide, a small-molecule treatment for RTT whose development was described above, costs around $500,000/year, depending on the patient’s weight. Ultimately, the commercial success of any future treatments for ASD/NDDs will depend on the assessment of the drug’s benefits and side effects by patients and their families, physicians, and payors as well.

Conclusion

We have seen major advances in genetic and developmental neurobiology research in ASD and related neurodevelopmental syndromes. In the case of RTT, these advances have already yielded an FDA-approved treatment that serves as an ideal case example of the process from gene identification to experiments in model systems to definitive clinical trials in human populations. Key elements that contributed to success in RTT included replicated findings for IGF-1 or its analogs in mouse models; a well-studied natural history that demonstrated developmental arrest without further improvement over time; development of RTT-specific outcome measures; and strong engagement of the RTT family community. Outside of rare genetic syndromes, attempts to target hypothesized mechanisms, such as excitatory-inhibitory imbalance, have not yet met with clear success in ASD, with limitations including statistical power, placebo response, and lack of a strategy to address heterogeneity.

We can draw some inferences from attempts to translate findings from the bench to the bedside in ASD and related NDD syndromes. At this point, most hypotheses carried into clinical trials have been based upon rodent studies, but less complex models may be better suited for small molecule screens. Molecular studies in human tissue, including iPSC-derived models and postmortem tissue, are critical to ensuring conservation across species. In contrast to RTT, success has proven more elusive in FraX, despite even stronger findings in mouse models, most likely because of differences in gene expression between rodents and primates (Kwan et al., 2012), which should emphasize the importance of cross-species comparison work. Importantly, FraX is also substantially different than RTT in many ways, including continued development that could masquerade as improvement during clinical trials.

When taking a new hypothesis into clinical trials, future studies should incorporate biomarkers and intermediate phenotypes to demonstrate target engagement in pilot samples and then extend these measures into large, well-powered studies. Much will remain uncertain about clinical trial design until there are FDA-approved interventions for core symptoms in ASD. When promising interventions enter phase 3 studies, we therefore should think not just about evaluating the treatments themselves but also about testing our methodology, potentially leveraging federal or philanthropic funding to add measures that extend beyond what is strictly required for potential FDA approval.

Footnotes

Authors’ Contributions

All authors contributed to the conceptualization, drafting, and editing of the article.

Disclosures

P.P.W. is an employee at Clinical Research Associates, LLC and a consultant for Neurogene, Roche, and Yamo Pharmaceuticals. In the past three years, J.V. has received research funding from Roche, Janssen, Acadia, Yamo, MapLight, and Clinical Research Associates, and has received an editorial stipend from Wiley. R.P.T. has received royalties from Oxford University Press and Springer Publishing.

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