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Consensus Guidelines for the Design and In Vitro Preclinical Efficacy Testing N-of-1


Antisense oligonucleotides (ASOs) can modulate pre-mRNA splicing. This offers therapeutic opportunities for numerous genetic diseases, often in a mutation-specific and sometimes even individual-specific manner. Developing therapeutic ASOs for as few as even a single patient has been shown feasible with the development of Milasen for an individual with Batten disease. Efforts to develop individualized ASOs for patients with different genetic diseases are ongoing globally. The N = 1 Collaborative (N1C) is an umbrella organization dedicated to supporting the nascent field of individualized medicine. N1C recently organized a workshop to discuss and advance standards for the rigorous design and testing of splice-switching ASOs. In this study, we present guidelines resulting from that meeting and the key recommendations: (1) dissemination of standardized experimental designs, (2) use of standardized reference ASOs, and (3) a commitment to data sharing and exchange.


Antisense oligonucleotides (ASOs) offer the unique opportunity for sequence-specific targeting of gene transcripts. This can be exploited to reduce expression of transcripts that give rise to toxic gain-of-function proteins via activation of RNase H, or to modulate RNA splicing to promote or restore expression of partially or fully functional proteins for diseases caused by haploinsufficiency or complete loss of proteins [1]. As such, ASOs offer potential treatment avenues for a plethora of common and rare genetic diseases, in particular those impacting the liver, central nervous system, and retina, tissues to which ASOs can be delivered efficiently. GalNac conjugates allow very robust ASO uptake by hepatocytes after systemic delivery, whereas local delivery is sufficient for broad uptake within the central nervous system and retina (via intrathecal or intravitreal injection, respectively) [2]. Although local treatment is invasive, a low rate of ASO turnover allows for relatively infrequent dosing: every 3–4 months for the central nervous system and 6 months for the eye.

Several oligonucleotides that require intravitreal or intrathecal delivery have received marketing authorization by the Food and Drug Administration (FDA) to treat eye and motor neuron diseases (fomivirsen, Macugen, and nusinersen) [1]. However, it has also demonstrated that it is possible to use ASOs to treat genetic mutations found in as few as an individual patient carrying a unique mutation within an academic setting [3]. Milasen, developed at Boston Children's Hospital, was a mutation-specific ASO for a child with Batten disease who carried an intronic mutation in the MFSD8 gene that resulted in cryptic splicing.

ASOs targeting the cryptic exon were designed and resulted in restoration of normal splicing and protein production when tested in patient-derived cell lines. After rat safety studies, an investigational new drug application (IND) was filed with the FDA, and investigational treatment with Milasen was initiated less than a year after discovering the pathogenic variant. Milasen administration was associated with a clear drop in the frequency and duration of epileptic seizures and a slower functional decline [3]. However, due to the late stage of her disease, Milasen could not reverse accumulated neuronal damage, and ultimately, the patient passed away 3 years later, although with improved quality of life.

Inspired by this example, initiatives such as n-Lorem were established [4] and also academic groups set out to develop N = 1 ASOs for individual patients such as the Dutch Center for RNA Therapeutics and the “1 Mutation 1 Medicine” (1M1M) initiative [5], and results of academic initiatives to develop individualized ASOs for FUS and C9orf72 amyotrophic lateral sclerosis have been published [6,7]. The N = 1 Collaborative (N1C) was set up as an umbrella organization to align and facilitate individualized treatment development efforts by sharing best practices and learning from successes and failures. N1C aims to provide tools for the different steps involved in N = 1 ASO development such as guidelines for patient/mutation selection, preclinical ASO design and testing, safety and toxicity tests, and regulatory aspects, as well as a toolkit to measure treatment effects in individual patients.

To develop consensus, interactive workshops on specific topics are organized by the N1C. Each workshop is attended by stakeholders involved in N = 1 ASO development: researchers in academia and industry, clinicians, foundations, and patients and their family members. While topics of specific workshops may appeal more to some stakeholders, N1C aims to solicit input from all stakeholder groups to inform and advance best practices for N = 1 ASO development.

This article is the result of an N1C workshop on ASO design and preclinical testing that was held online on July 25, 2022. The workshop covered both exon skipping and RNase H ASOs with the goal of advancing standards for development that are simultaneously rigorous and efficient (reflecting the clinical needs of patients involved in these early efforts, who often have rapidly progressive diseases for which “time is neurons”). This article will focus on the design of exon skipping ASOs, while an accompanying forthcoming article will focus on RNase H ASOs.

Annemieke Aartsma-Rus, Alejandro Garanto, Willeke van Roon-Mom, Erin M. McConnell, Victoria Suslovitch, Winston X. Yan, Jonathan K. Watts, Timothy W. Yu, and on behalf of the N = 1 Collaborative.Consensus Guidelines for the Design and In Vitro Preclinical Efficacy Testing N-of-1 Exon Skipping Antisense Oligonucleotides.Nucleic Acid Therapeutics.Feb 2023.17-25.


We envision a future where individualized medicine centers around the world routinely offer patients customized treatments targeting their condition’s underlying genetic cause.



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