In just one year in 2018, the US saw the development of Milasen (Boston Children’s Hospital), a patient-customized ASO, by a Boston-based team for 6-year-old Mila Makovec with neuronal ceroid lipofuscinosis 7 (CLN7), a form of Batten’s disease [34]. The condition was attributed to a unique cryptic splicing variant in the CLN7 gene (Table 1). This pioneering effort paved the way for additional N-of-1 or N-of-few ASOs tailored for individual or a handful of patients using expedited regulatory pathways. [35]

For instance, Atipeksen (Boston Children’s Hospital), an individualized ASO designed for a specific mutation causing ataxia-telangiectasia (AT) [36], initiated at an early age in a 3-year-old patient. Notably, this treatment started before the typical onset of neurological symptoms (typically at age 6 or 7), showcasing the potential to preserve motor function effectively.

Furthermore, the development of Afinersen (RNA Therapeutics Institute, UMass Chan Medical School, Massachusetts) targeting Amyotrophic Lateral Sclerosis (ALS) with a C9ORF72 mutation underwent a single-patient pilot study in 2019 in a 60-year-old man, following successful animal model testing. This suggests potential applicability to other patients with the same gene mutation [37]. Jacifusen (Ionis Pharmaceuticals, California), an ASO tailored for a particular mutation in FUS causing ALS, demonstrated promising efficacy in preclinical studies and subsequent patient applications [38]. Jaci Hermstad, aged 25, was the pioneering recipient of this treatment, diagnosed in February 2019, coinciding with the ongoing development of Jacifusen by Ionis (formerly known as ION363). Subsequent to Jaci's treatment, numerous other patients received the therapy, with approximately 12 IND applications submitted for the same therapeutic. This substantial interest culminated in a comprehensive clinical trial sponsored by Ionis (NCT04768972), marking a pivotal shift from individual case studies to broader clinical applications. [39]

Currently, many N-of-1 treatments are focused on brain and eye diseases (Table 1) due to the relative accessibility of these tissues and the challenges associated with targeting other tissues, such as muscles. While Table 1 provides examples of these innovative approaches, it is important to note that this is not an exhaustive list, as numerous other treatments and programs are also underway, reflecting the breadth of advancements in the field.

However, alongside these success stories, setbacks have been encountered, as illustrated by the ASO Valeriasen (Boston Children’s Hospital), underscoring the complexities inherent in personalized treatments. In 2020, two toddlers with a severe form of epilepsy, both harboring a KCNT1 mutation, were treated with the N-of-few therapy. While the drug reduced seizures in one patient and eliminated them in the other, both developed hydrocephalus, resulting in the cessation of treatment [40]. Although one patient's death was unrelated, the trial was paused, pending FDA approval to resume therapy for the second patient at a lower dose, with investigations underway to mitigate hydrocephalus risk. Similar occurrences of hydrocephalus have been noted with ASOs for other conditions, such as Tominersen (Genentech, California) for Huntington’s disease [41] and Nusinersen (Biogen, USA) for spinal muscular atrophy [42], although establishing causation proves challenging due to the multifaceted nature of neurologic diseases and varying ASO characteristics. Such setbacks emphasize the importance of thorough risk assessment and continuous monitoring in the pursuit of personalized therapies.

In Europe, ASO examples include, among others, ongoing trials for PLP1 mutation-induced hypomyelination of early myelinating structures and trials for Atipeksen, and the development of an individualized therapy by Institut Imagine for a patient with a KCNB1 gene mutation [43], highlighting the global momentum in advancing tailored treatments for rare genetic mutations. [44, 45]

In addition to ASO-based approaches, CRISPR gene editing has emerged as a potential N-of-1 and N-of-few therapy. CRISPR technology, unlike RNA-based therapies, directly edits the DNA to correct genetic mutations, offering a more permanent solution. A notable example is CRD-TMH-001 developed by Cure Rare Disease (Boston, Massachusetts) for Duchenne muscular dystrophy (DMD) [46]. This therapy, targeting muscle promoter and exon 1 mutations on the dystrophin gene, entered a clinical trial (NCT05514249) in 2022 for a patient with DMD. Unfortunately, the patient's health deteriorated rapidly six days after receiving the therapy, displaying cardiac and respiratory distress that led to their death. Post-mortem findings revealed lung injury due to a heightened immune response to the high-dose AAV vector [47]. The absence of Cas9 expression in the patient's body indicated the therapy remained inactive. However, this highlighted the potential strong toxic effect of 'first generation' vectors like AAV9 when delivered at high doses needed for widespread reach to muscles, particularly in patients at advanced disease stages with weakened physical conditions.

The main challenge in N-of-1 CRISPR cures is the irreversible nature of gene editing compared to the adjustability of ASO dosing. Balancing accelerated de-risking and patient safety is crucial. While significant investments can be justified for chronic severe diseases with large patient pools reaching hundreds of thousands, the exorbitant costs (up to $5 million) associated with N-of-1 CRISPR for unique cases, where the window for life-saving editing is narrow, render this approach currently impractical.