Applying Synthetic Nucleic Acid Therapeutics to Gene Activation for Haploinsufficient Diseases

Abstract

Haploinsufficient autosomal dominant diseases are due to heterozygous mutations that cause inadequate protein expression. Compounds that increase expression of the wild-type allele would be one strategy for treating patients. Synthetic antisense oligonucleotides and double-stranded RNAs have the potential to increase gene expression, making them starting points for drug development. Our goal is to outline strategies for using synthetic nucleic acids to enhance gene expression. We discuss the strengths and limitations of these strategies and the practical challenges behind upregulating the expression of genes as a treatment for haploinsufficient autosomal dominant diseases.

Keywords

haploinsufficient autosomal dominant disease antisense oligonucleotide double-stranded RNA gene activation splicing untranslated regions

Introduction

Synthetic antisense oligonucleotides (ASOs) and double-stranded RNAs (dsRNAs) are now an established strategy for drug development.^1,2 As of 2025, there are at least 13 ASOs and 6 dsRNAs that have been approved by the Food and Drug Administration (FDA).^3,4 These new drugs are having major positive impacts on the lives of patients, and there will probably be several additional drug approvals over the next 5 years. Most nucleic acid therapies use gapmer ASOs or silencing dsRNAs (siRNAs) that reduce the expression of a gene involved in disease. For some diseases, however, compounds that increase gene expression would be beneficial.

Haploinsufficient diseases are genetic conditions caused by mutation of one allele of a gene resulting in inadequate protein production (Table 1). Haploinsufficient diseases are typically autosomal dominant. They occur when the mutant gene produces no protein at all, too little protein, an inactive protein, or a protein that causes a phenotype directly by disrupting an important cellular pathway.^23,24 These mutations can be inherited but will more often arise sporadically.

Table 1.

A List of 10 Examples of Haploinsufficient Autosomal Dominant Diseases Without an Effective Therapy^5–22

Haploinsufficient disease	Gene(s) affected	Approximate number of diagnosed patients to date	Current treatment approach
Autosomal dominant polycystic kidney disease (ADPKD)	Polycystin 1 (PKD1)	600,000 ∼ 12,500,000	Hypertension medication, dialysis, kidney transplant
Autosomal dominant polycystic kidney disease (ADPKD)	Polycystin 2 (PKD2)	600,000 ∼ 12,500,000	Hypertension medication, dialysis, kidney transplant
Tetralogy of Fallot (TOF)	Neurogenic locus notch homolog protein 1 (NOTCH1)	1,848 ∼ 4,781	Beta-blockers, device implant, heart transplant surgery
Tetralogy of Fallot (TOF)	Fms-related tyrosine kinase 4 (FLT4)	1,848 ∼ 4,781	Beta-blockers, device implant, heart transplant surgery
Phelan-McDermid syndrome (PMS)	SH3 and multiple ankyrin repeat domains 3 (SHANK3)	∼3,100 ∼ 3,600	Anti-seizure medications, device implantation, rehabilitation
Margan syndrome (MFS)	Fibrillin 1 (FBN1)	412 ∼ 2,329	Beta-blockers, angiotensin receptor blockers
Pitt–Hopkins syndrome (PTHS)	Transcription factor 4 (TCF4)	∼500 ∼ 1,518	Anti-seizure medications, rehabilitation
Stickler syndrome	Collagen gene family (COL2A1, COL9A1/2/3, COL11A1/2)	500 ∼ 999	Device implant, rehabilitation, surgery
Osteogenesis imperfecta (OI)	Collagen gene family (COL1A1/2)	∼724, 959	Biphosphonate and hormone treatments, device implant, rehabilitation
Heterozygous familial hypercholesterolemia (HeFH)	LDL receptor (LDL-R)	105 ∼ 572	Statins
	Apolipoprotein B (APOB)
	Proprotein convertase subtilisin/kexin 9 (PCSK9)
SLC6A1-related disorders	Solute carrier family 6 member 1 (SLC6A1)	116 ∼ 500	Antiseizure medications, rehabilitation
Early-onset Macular Drusen (EOMD)	Complement factor H (CFH)	>89	N/A

For each disease, the following are indicated: (1) the affected gene(s) that are linked to the disease pathology, (2) the approximate number of patients diagnosed with the disease based on current literature, and (3) the current approaches to treat disease symptoms.

This perspective will focus on gene activation as a strategy to treat haploinsufficient autosomal dominant genetic disease (Table 1).^5–22 We will first describe an experimental pipeline for assaying gene activation by synthetic ASOs and dsRNAs. We will then describe strategies to promote gene activation and restore protein function.

Setting up an Experimental Pipeline

Identify experimental models

The first step toward developing an experimental pipeline for compound screening is to identify a cell culture model that adequately expresses a gene-of-interest (Fig. 1A). To achieve this first step, we recommend using the Human Protein Atlas database, which surveys gene expression across human tissues and commercial cell lines.^26,27 The chosen cell line may be a patient-derived line, which is ideal due to its physiological and clinical relevance in translating the success of promising compounds identified in culture. However, patient-derived cell lines may not always be available. If they are needed, it may be necessary to derive them from patient cells. In the absence of patient cells, the accessible availability of commercial cell lines that express the gene-of-interest will therefore be an advantageous and critical starting point.

FIG. 1.

Setting up an experimental workflow and pipeline to screen synthetic nucleic acids. (A) Select a relevant, appropriate cell model. Cells could be derived from patients and carry the mutant allele. Alternatively, cells could have wild-type alleles. (B) Establish transfection or electroporation conditions. The following panel illustrates gene activation resulting from on-target effects. Quantitate on-target effects by appropriately designed synthetic nucleic acid controls using RT-qPCR and western assays.²⁵ The goal is to develop robust assays that can discriminate small on-target effects from confounding off-target effects. (C) Characterize the DNA and RNA landscapes for a gene-of-interest, identifying targets to prioritize the design of complementary synthetic ASOs or dsRNAs. ASOs, antisense oligonucleotides; dsRNAs, double-stranded RNAs.

A wild-type cell line might be adequate if the goal is to enhance expression to a level closer to normal, and wild-type cells will often be readily available. A mutant cell line would be necessary if the strategy involves correcting the expression of a mutant gene. Whichever model is chosen, the cells should be straightforward to grow and manipulate because robust testing is necessary to demonstrate that activation can be achieved by an “on-target” mechanism.

In some cases, however, there may not be an available cell line that robustly expresses a gene-of-interest. Minigene plasmid reporters can address this limitation. Using a cell line that is easy to grow, minigenes can be transfected into these cells to express the gene-of-interest. From the promoter driving gene expression, to the specific sequence context for a gene-of-interest, minigenes are customizable to meet the evolving needs of a project. For example, if a patient-derived line is not available, minigenes can be advantageous for characterizing mutation-induced splicing defects and screening for ASOs that can correct these defects in a disease context.²⁸

After identifying a promising candidate compound in cell culture models, animal testing may be the next step. If there is a good animal model available, animal studies can be used to support the conclusion that the compounds have the ability to correct a disease phenotype, increasing confidence that clinical trials are justified. Unfortunately, good animal models may not always be available, or the details of gene regulation or physiology may differ significantly between mice and humans. The lack of a good animal model should not derail drug development efforts but will need to be discussed at an early stage with regulatory authorities.

For sporadic rare diseases, there might not be the time or the resources necessary to develop an animal model when the needs of patients are immediate. The lack of a good animal model would make thorough testing in cell culture even more important. In that case, it will be helpful to have a cell culture model that mimics elements of the functional phenotype. For example, for a disease that is characterized by splicing defects in affected gene(s), synthetic nucleic acids that reverse these defects would serve to reassure that it is possible to intervene through the predicted molecular mechanism beyond cell culture models.

Establish that effects are efficient and on-target

Once a cell culture model is chosen, it is necessary to develop protocols for efficient introduction (ie, transfection, electroporation, gymnotic delivery, etc.) of synthetic nucleic acids into that cell line (Fig. 1B). It is important to have the reagents necessary for quantitative analyses, including primer sets for quantitative PCR (qPCR) and antibodies for western blotting. The availability of an adequate commercial antibody against the protein-of-interest would be an asset, but researchers may find that it is necessary to obtain and validate an adequate antibody themselves. All commercial antibodies should be carefully validated, as these do not always function as advertised.

The first step for compound testing is to identify a positive control ASO or dsRNA that can knock down the expression of the target gene or surrogate benchmark gene, relative to an appropriate negative control such as a noncomplementary ASO or dsRNA. Positive controls will establish that the introduction of active synthetic nucleic acids into cells has been achieved.

Negative controls can include mismatched and scrambled sequences based on positive controls or based on any candidate sequences that are uncovered during the screening for activating compounds. One can never have too many controls nor too many replicates. While use of phosphate buffered saline (PBS) alone is a worthy comparison (PBS is the solution many ASOs and dsRNAs are dissolved in prior to in vivo administration), experimenters should be careful before making it the only comparison because the absence of a similarly designed nucleic acid negative control will not make for an “apples-to-apples” comparison with demonstrate “on-target” effects by a positive control or active compounds.

The combination of positive and negative controls establishes that an “on-target” effect has been observed. While no set of controls are perfect, inadequate use of controls during relatively inexpensive cell culture testing may lead to misleading data and a wasted investment as projects move to more expensive animal or preclinical testing. Building a strong foundation to support an “on-target” mechanism in cell culture is essential. A clear description of controls and why their use supports the conclusion that a positive result is likely due to “on-target” engagement should be a central part of any presentation of results.

Why are controls so important? The general need for controls when discovering promising synthetic nucleic acid compounds was first documented as far back as the 1990s.²⁵ More recently, updated guidelines have described in detail the motivation behind the need for controls and descriptions of the types of controls that should be used.²⁵ Rigorous adherence to these guidelines is essential to avoid wasting resources on the development of candidates with obvious flaws. For example, a 2.0-fold increase in gene expression that could restore protein levels is relatively small. Establishing that a small change is an on-target effect will always be challenging. A small change in expression of a reference gene might cause a misleading qPCR result. In cell culture, routine variations in cell confluence or serum conditions might affect gene expression and result in false positives. Replicate experiments and controls are the only path to building a solid foundation for subsequent discovery and development. We will return to this discussion below in the subsection Evaluating gene activation.

Understand DNA and RNA landscapes

It is important to understand the “DNA and RNA landscapes” that characterize target genes (Fig. 1C). A DNA and RNA landscape includes data on both structure and function of gene sequences. These data will help guide the design of synthetic nucleic acids intended to activate gene expression. Understanding the DNA and RNA landscape requires thorough characterization of transcription at a gene locus. Where does transcription of mRNA begin? Where does it end? What noncoding RNA transcripts, if any, are present that overlap the gene? A full description of DNA and RNA landscape characterization would require its own Perspective; our goal is to introduce this topic.

The first step is to compile information from databases. We first use the Cap Analysis of Gene Expression sequencing (CAGE-seq) data from the Functional ANnoTation Of the Mammalian genome (FANTOM) consortium to identify and rank transcription start sites.²⁹ We then use the Encyclopedia of DNA Elements (ENCODE) database³⁰ to complement this information with information on: (1) ChIP-seq analysis of transcription factors, (2) ChIP-seq analysis of histone markers, and (3) DNase-seq analysis of chromatin accessibility. Eukaryotic Promoter Database New (EPDnew)³¹ is a prediction tool that can supplement identification of transcription start sites.

Understanding the RNA landscape includes identifying interactions between RNA and critical protein partners. Association of protein partners can be estimated using computational tools like RBPmap to scan a given sequence and predict binding sites for specific RNA-binding proteins (RBPs).³² Crosslinking immunoprecipitation (CLIP) is a technique used to define RNA:protein associations.³³ Publicly available CLIP datasets can offer experimental support to complement predictions.

While information from databases is useful for initial planning, it will often be worthwhile to experimentally characterize the RNA and DNA landscapes of a gene-of-interest in the cell line that will be used to screen compounds. To characterize the DNA landscape, assays can include: (1) 5′ rapid amplification of cDNA ends (RACE) to identify transcription start sites, (2) ChIP-qPCR analysis of transcription factors and/or RNA polymerase II binding, (3) DNase hypersensitivity analysis to interrogate chromatin structure, or a (4) simple qPCR assay designed to “walk” across a putative promoter region with different qPCR primers to establish a gene expression profile of where a gene starts and the relative efficiency of different start sites. While expression of genes that are under regulation of TATA-boxes may be relatively simple, genes with TATA-less promoters can have much more complex promoter landscapes.

To characterize the RNA landscape, assays include: (1) 5′ or 3′ RACE to determine the boundaries of coding and noncoding transcripts, and (2) CLIP-based approaches to identify binding sites for specific RBPs. For example, anti-AGO2 chimeric eCLIP is a recent variant of eCLIP that identifies miRNA:RNA interaction sites,³⁴ allowing more efficient prioritization of potential targets involved in regulation by miRNAs and more efficient use of resources needed for rigorous experimental validation. Given the stakes of these projects and the relatively low cost of modern sequencing methods, experimental characterization in the most disease-relevant cell lines available will almost always be a good investment. Even if such characterization does not directly lead to the design of an active compound, thorough understanding of regulatory landscapes will offer the potential for productive hypotheses and unexpected benefits during development.

Evaluating gene activation

Once cell lines, assay protocols, and working hypotheses for nucleic acid designs are in hand, testing for gene activation can begin. In some circumstances, target sequences will be obvious, and a few synthetic nucleic acids will be sufficient. In most cases, however, a larger series of ASOs or dsRNAs will need to be screened. If replicate screening reveals reproducible activation by one or more potential activating compounds, those lead compounds will then need to be pursued in depth.

An autosomal dominant, haploinsufficient mutation will result in, at most, a 50% loss of protein production. A useful therapeutic intervention may yield a 2.0-fold increase in gene expression, but even a smaller amount might be beneficial. It may be difficult to distinguish signal from noise, and all promising synthetic nucleic acids face the same initial challenge is an observed activity the result of on-target gene regulation due to a physical interaction at the intended target site, or an off-target effect?²⁵ Experimenters should decide on convincing statistical tests and requirements for replicating data before collecting data.

Introducing synthetic nucleic acids into cells can be a messy business. If one tests enough ASOs or dsRNAs, one is almost assured to identify one or more compounds that seem to increase gene expression by 1.5 to 2.0-fold, exactly the type of increase that would constitute success for a program that aims to achieve activation of a haploinsufficient gene. Such small increases can often be attributed to off-target effects that have nothing to do with engagement of the gene-of-interest.

Fortunately, the use of ASOs and dsRNAs are well-adapted to control experiments.²⁵ It is essential that rigorous control experiments and skepticism about “positive” experimental results be an integral part of every step of a development program. After identifying a promising compound, the most important step in any discovery campaign will be the ruthless investigation of alternate off-target explanations for the observed gene activation.

Oligonucleotide Chemistry

Over 40 years of innovation has produced a well-stocked toolbox of chemical options for improving the properties of synthetic nucleic acids.^1,2 This impressive selection has been described recently and will not be described in detail here. However, examples of important chemical modifications include phosphorothioate linkages. These linkages stabilize synthetic nucleic acids against digestion by nucleases and can favorably affect biodistribution. 2′-modifications, like 2′-fluoro, 2′-O-methyl, and 2′-O-methoxyethyl can additionally stabilize synthetic nucleic acids against nuclease digestion. The 2′-modifications also lead to tighter binding with target sequences. Synthetic nucleic acids can also be conjugated to include targeting ligands, like N-acetylgalactosamine, which have led to breakthrough increases in effective delivery in vivo through binding to cell surface receptors. The purpose of these modifications is to optimize delivery to target tissues, increase potency, reduce “off target” effects on the expression of nontarget genes, and, ultimately, create the widest therapeutic window between efficacy and side effects for patients.

As the field advances, exciting new innovations will become available over the next 5–10 years to further enhance the power of synthetic nucleic acids. While these advances will likely have major impacts, in the near term, the more established approaches used in successful development efforts will better starting points (Fig. 2). For example, the first choice for ASOs designed to modulate splicing might be phosphorothioate methoxyethyl oligonucleotides inspired by Spinraza. dsRNAs may use the same chemical modification scheme or conjugation strategies as those used for current drugs and advanced clinical trials. Patients with rare diseases need results quickly, and the toolbox of “off the shelf” chemistries offers many options for lead compound identification.

FIG. 2.

A select list of FDA-approved, commercially available synthetic nucleic acids.^1,2 For each compound, the following are indicated: (1) the targeted disease, (2) the gene and its respective region targeted, (3) the mechanism-of-action by which the compound acts to be therapeutically beneficial, and (4) the sequence and chemistry of the compound. The chemistry used in each compound is annotated at the bottom of the figure.

Researchers are encouraged to avoid “reinventing the wheel,” not only with regard to chemistry but also methodology. Many members of the oligonucleotide therapeutics field have a reputation for offering generous insights. New entrants to the field are encouraged to seek out this advice to identify the most efficient path toward testing their hypotheses.

Mechanisms

Activating gene expression by targeting splicing regulatory sequences

ASOs that modulate splicing are an established method for upregulating the expression of full-length, mature mRNAs that code for functional protein (Fig. 3A).^35,36,40,41 An example is Spinraza for treating spinal muscular atrophy (SMA). SMA is not a haploinsufficient autosomal dominant disease, but its clinical success highlights the therapeutic power of synthetic nucleic acids and the value of modulating splicing to upregulate gene expression.

FIG. 3.

Targeting pre-mRNA splicing to activate gene expression. The models shown in (A) and (B) are examples of mechanisms for gene activation that rely on changes in splicing. (A) Modulation of pre-mRNA splicing by targeting splicing regulatory sequences with synthetic nucleic acids.^35,36 (B) Modulation of pre-mRNA splicing by targeting alternative splicing events that induce nonsense-mediated decay.^37–39

SMA is an autosomal recessive disease caused by mutations in both alleles of the Survival Motor Neuron 1 (SMN1) gene.⁴² These mutations lead to a deficiency of full-length, functional SMN proteins necessary for neuromuscular functions. A paralog of SMN1, SMN2, remains unaffected in SMA. However, the SMN2 gene has a single nucleotide change from SMN1 that results in less efficient splicing of exon 7, producing an unstable SMN protein variant. The loss of SMN1 and the inability of SMN2 to compensate for the overall loss of stable SMN proteins are the cause of SMA.

Analysis of SMN2 splicing patterns suggested that an ASO that increased splicing of exon 7 might produce a more stable SMN2 variant protein and increase overall SMN function.^43,44 This effort led to Spinraza (Nusinersen),^45–47 a methoxyethyl phosphorothioate ASO that improves the lives of SMA patients. Spinraza works by blocking a repressor of exon 7 splicing to enhance SMN2 exon 7 splicing and full-length SMN protein production. The development of Spinraza, however, had some unique advantages. Most development programs will not be able to exploit the “activate the spare gene” strategy to upregulate gene expression. Thorough characterization of associated regulatory pathways that can influence splicing mechanisms will be necessary to develop splice-modulating ASO to activate gene expression. As with seeking advice about nucleic acid chemistry from experts, projects that seek to manipulate splicing should incorporate advise from researchers with broad knowledge of splicing mechanisms.

Activating gene expression by targeting “poison” exons that induce nonsense-mediated decay

Approximately 95% of genes undergo alternative splicing (AS) events that can lead to the inclusion of alternative exons or the usage of alternative splice sites.⁴⁸ At least 33% of AS events can introduce “poison exons,” which contain premature stop codons that trigger the nonsense-mediated decay (NMD) pathway to degrade these nonproductive transcripts.⁴⁹ Modulating AS events that induce NMD, therefore, are another approach to upregulate gene expression (Fig. 3B).

Stoke Therapeutics and collaborators have established a powerful target discovery pipeline to identify AS events in various rare disease contexts called targeted augmentation of nuclear gene output (TANGO).³⁷ The application of TANGO led to the discovery of ASOs that can induce skipping of a poison exon within the SCN1A gene, leading to upregulated SCN1A mRNA and protein expression in human cells and mice models.³⁸ This effort by Stoke Therapeutics led to the ASO drug Zorevunersen for Dravet syndrome, an autosomal dominant haploinsufficient disease. Zorevunersen recently received the Breakthrough Therapy Designation from the FDA and is currently in Phase 3 clinical trials.³⁹

Blocking miRNA-mediated inhibition of translation

miRNAs are small endogenous RNAs that are best known to bind within the 3′ untranslated region (UTR) of genes to inhibit gene expression.^50–55 The repression of gene expression by miRNAs can be reversed by an ASO (ie, “antagomirs”) that targets the miRNA primarily responsible for repressing gene expression,⁵⁶ or by an ASO that blocks the binding site for that miRNA.⁵⁷ If the gene responsible for a haploinsufficient disease is repressed by a miRNA, an ASO that blocks the action of the miRNA is therefore a logical strategy for therapeutic development.

Unfortunately, this attractive hypothesis faces challenges. Regulation by miRNAs cannot be assumed, and the involvement of miRNA mechanisms must be carefully established.⁵⁴ While modern experimental tools and improved understanding of mechanisms allow the potential impact of miRNAs to be estimated with higher accuracy than previously possible,^34,58 experimental validation of miRNA-mediated control remains challenging. For many genes, miRNAs may have no impact on expression. Even for those genes that show evidence for the association of miRNAs, that association may affect gene expression too little to be a vulnerability that can be exploited to increase gene expression. As such, caution and rigor are required if one seeks to target miRNA mechanisms with synthetic nucleic acids. To end this section on a more positive note, if a miRNA mechanism is implicated to repress gene expression, blocking miRNA-mediated repression may be one of the simplest paths toward upregulating expression of a wild-type gene and discovering a potential compound that can compensate for a haploinsufficiency.

Activating gene expression by targeting the 5′ or 3′ UTR

The translation and stability of an mRNA is dictated by information within the 5′ and 3′ UTRs of genes. The 5′ UTR can contain multiple open upstream reading frames (uORF) upstream of the primary uORF, determining the translation efficiency of an mRNA.⁵⁹ Designing synthetic nucleic acids to stably perturb uORFs can increase protein levels in a dose-dependent manner (Fig. 4A).⁶⁰ This strategy to target the uORFs of mRNAs is a sequence-specific approach that may broadly activate gene expression. Conversely, the 3′ UTR of mRNA can poise a gene’s expression to be silenced through miRNA-mediated control. One strategy to activate gene expression could be to design synthetic nucleic acids that compete with miRNAs validated to bind the 3′ UTR, preventing gene silencing by an inhibitory miRNA (Fig. 4B).⁵⁷ Simultaneously targeting the 5′ and 3′ UTR of an mRNA with synthetic nucleic acids can also prove effective in stabilizing transcripts to enhance overall gene expression.⁶¹

FIG. 4.

Targeting the 5′ or 3′ untranslated regions to activate gene expression. (A) Modulation of mRNA translation by targeting upstream open reading frames in the 5′ UTR with synthetic nucleic acids.⁶⁰ (B) Modulation of mRNA translation by targeting miRNA binding sites in the 3′ UTR with synthetic nucleic acids.^57,61

Activating gene expression by targeting promoter regions

Noncoding RNA transcripts that overlap gene promoters can inhibit gene expression (Fig. 5).⁶³ If an AGO:RNA complex binds to a non-coding RNA transcript while it is in proximity to the promoter, it can act like a ribonucleoprotein transcription factor to modulate gene expression. Depending on the position of binding, synthetic nucleic acids like dsRNAs can associate with AGO to bind noncoding RNA transcripts that overlap gene promoters and activate gene expression.^64–69 This mechanism of gene activation has the potential to upregulate the wild-type copy of a haploinsufficient gene and correct the haploinsufficiency. The leading hypothesis is that this mechanism involves cis interactions with nascent transcripts that are associated with the chromosomal locus rather than a trans interaction with a transcript that is made elsewhere. Cis interactions would indicate the participation of rare transcripts (one or two copies per cell).

FIG. 5.

Targeting promoter-associated, non-coding RNA transcripts to activate gene expression. An AGO:small RNA complex binds to a nascent transcript at a gene promoter.⁶² The bound AGO:RNA complex has the potential to act as a ribonucleoprotein transcription factor to up-regulate gene expression.

The mechanism of promoter-targeted RNA has been reviewed recently and will not be described in detail here.⁶³ From a practical standpoint, the first task is to examine the gene promoter and identify the transcription start site. The next step is to identify any transcription of a noncoding RNA transcript that might overlap the promoter of a target gene. Expression of the noncoding RNA transcript does not need to be as high as the mRNA but should be at least one copy per cell. If experiments confirm the identity of a promoter-associated non-coding RNA, the next step is to target dsRNAs to sequences within 100–200 bases of the transcription start site and assay for gene activation.

Future Perspectives

The stakes of drug development for rare diseases are high. Progress should be as rapid as possible since the condition of patients may worsen over time. ASOs and dsRNAs have shown promise as N = 1 therapies because of their inherent advantages for rapid development.^70–72 Further, patient-derived organoids can now be generated rapidly, offering unprecedented power and speed to evaluate the therapeutic potential of an ASO or dsRNA in a relevant disease and physiological context.⁷³ These advantages, combined with rigor and the use of past experiences to avoid mistakes, make synthetic nucleic acids an important alternative for the development of agents to treat haploinsufficient diseases.

As a field, oligonucleotide therapeutics are achieving a level of broad success that cuts across chemical modality, disease type, and conjugate design. For example, delivery to target tissues has been and continues to be a challenge, but promising advances are being made to many tissues, including the central nervous system and muscle. No one could have anticipated the rapid pace of improvement when the first trials of Spinraza were starting.

This accelerating success justifies hope that synthetic oligonucleotides will achieve more success treating rare diseases. It will be important, however, that patients, families, and their advocates share information and research materials. The need for progress is urgent, and the many lessons learned in one disease will apply to others. The family-led foundations should find common ground to speed drug discovery and development. For scientists and industry working in the field, the free sharing of material and knowledge (and this is often the case now) should become even more routine. The field has a great base to build upon, and the opportunities for disciplined, knowledge-driven, collegial science using synthetic oligonucleotides have never been more promising.

Conclusions

There are several different strategies to activate gene expression to alleviate haploinsufficient disease. Regardless of which strategy is chosen as a priority, all strategies share more similarities than differences. They all involve synthetic nucleic acids, similar transfection protocols, and similar assays to determine gene expression. Because of these similarities, testing multiple strategies for gene upregulation is feasible but may require only an incremental increase in effort beyond testing the priority strategy alone. Given the stakes involved for patients, the modest investment required in surveying different modalities in the same cell culture models with the same assays may be justified. Drug candidates that may show the most promise are likely those that draw on the experience and design of previously approved ASOs and dsRNAs, reducing the barriers to developing treatments for patients with continued unmet needs.

Footnotes

Acknowledgments

The authors acknowledge with gratitude support provided by the Vincent Pauca “Be Better” Memorial Award from the Pitt Hopkins Research Foundation. The authors thank Bethany Janowski for her critical feedback on this article.

Author Disclosure Statement

D.R.C. is a founding scientist at Iris Medicine.

Funding Information

D.R.C. was supported by the National Institutes of Health (NIH) (GM R35GM118103) and the Robert Welch Foundation (I-2184) and holds the Rusty Kelley Professorship in Medical Science.

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