Table 3.

Pros and Cons of Each TNA Strategy

Therapeutic strategies	Advantages	Disadvantages	Refs
ASOs	- Easy to synthesize, lower cost - Different modes of action: transcript cleavage, steric blocking - Sufficient intracellular uptake through endocytosis without an additional delivery vehicle (all of 7 FDA- approved ASOs were not designed to associate with a delivery agent) - Low immunoreactivity - Excellent target specificity, capacity toward ‘small molecule or protein-undruggable targets’ - Advances in chemical modifications of the backbone and nucleotides increase stability from nuclease degradation and reduce toxicity - Rapid development, timely production, especially for patient-customized ASOs - Have a long history of clinical development. There have been 8 approved ASOs since 1998, demonstrating safety of ASO drugs	- Unmodified ASOs are less stable than siRNAs - Main concerns for effective clinical applications include stability to nuclease degradation, delivery, off-target events. - In many cases, approved ASOs are extremely expensive - Accumulate in the highest concentration in liver and kidney - Host gene targeting-ASOs may cause adverse effects through affecting endogenous biological functions involved target genes	[9,83,85., 86., 87., 88., 89., 90.]
siRNAs	- Natural duplex siRNAs are more stable than ASOs - High target specificity, capacity toward small molecule or protein-undruggable targets - Chemical modifications have been developed to enhance the stability and activity and reduce immune stimulation and toxicity as well - Conjugation with a ligand and/or encapsulation with a nanocarrier such as LNP enhances delivery - Two approved siRNAs including patisiran (LNPa formulation) and givosiran (GalNAc conjugate)	- More expensive production than ASOs - Poor intracellular uptake without delivery aids - Main problems for their effective clinical application involve delivery, stability, and off-target effects - Inducing the innate immune responses by siRNA itself and its associated delivery vehicles - Accumulate in the highest concentration in liver and kidney	[9,83,85,91., 92.]
CRISPR/Cas	- Powerful tool for gene editing and precision medicine - Achieve both DNA and RNA targeting - The use of synthetic gRNAs enables incorporation of chemical modifications, avoiding nuclease degradation and cellular toxicity - Nonviral delivery methods (such as LNP) can facilitate transient Cas expression, reduce off-target effects, lower immune activation, and achieve tissue-specific targeting	- More expensive production than ASOs - Inducing immune stimulation (both innate and adaptive immune responses), poor intracellular uptake, off-target events - Adaptive immune activation and even pre-existing immune responses in human against Cas9 - Delivery of both gRNAs and Cas (4-kb gene) are crucially required - Viral delivery methods have limited packaging capacity, high immunogenic potential (especially for repeated doses), high frequency of off-target events (due to longevity of Cas expression)	[93., 94., 95., 96.]
mRNA vaccines	- Safety, tolerance, and high potency - Rapid development, ease of production, and scalability - Formulating mRNA into nanocarriers such as LNP enhances efficient in vivo delivery - Safer profile than DNA vaccine. Low risk of oncogenic potential due to genomic integration as for DNA vaccines - Availability of numerous technologies can be applied to enhance stability and translation rate and reduce immunostimulatory effects of mRNA vaccines, such as: + The use of modified nucleosides reduces innate immune activations + Optimization of poly(A) tail, regulatory elements sequence, and/or codon optimization to enhance stability and translation rate + Self-amplifying RNA (saRNA) vaccines induce immune responses with considerably lower mRNA inputs	- Naked mRNA vaccine is sensitive to nuclease degradation and exhibits poor intracellular uptake - Immunogenicity - Deep-frozen storage is required - New technology (there has not been any approved mRNA vaccine so far). Need to establish safety regulations and legislation	[67,69., 70., 71.,97., 98., 99.]

^aLipid nanoparticle.