Nucleic acid therapeutics最新文献

The recent Nobel Prizes awarded to Ambros and Ruvkun have refocused attention on microRNAs (miRNAs). The importance of miRNAs for basic science has always been clear, but the application to therapy has lagged behind. This delay has been made even more apparent by the accelerating pace of successful programs using duplex RNAs and antisense oligonucleotides to target mRNA. Why has progress been slow? A clear understanding of how miRNAs function in mammalian cells is obscured by the fact that miRNAs can exert their effects through multiple complex mechanisms. This gap in our knowledge has complicated progress in drug discovery. Better insights into the mechanism of miRNAs, more rigorous definitions of miRNAs, and more powerful tools for establishing the physical contacts necessary for miRNA action are now available. These advances lead to a central question for nucleic acid therapy-can miRNAs be productive targets for drug discovery and development?

Antisense oligonucleotides (ASO) are very promising drugs for numerous diseases including neuromuscular disorders such as Duchenne muscular dystrophy (DMD). Several ASO drugs have already been approved by the US Food and Drug Administration for DMD and global efforts are still ongoing to improve further their potency, notably by developing new delivery systems or alternative chemistries. In this context, a recent study investigated the potential of different chemically modified ASO to induce exon-skipping in mouse models of DMD. Importantly, the authors reported a strong discrepancy between exon-skipping and protein restoration levels, which was mainly owing to the high affinity of locked nucleic acid (LNA) modifications to the target RNA, thereby interfering with the amplification of the unskipped product and resulting in artificial overamplification of the exon-skipped product. These findings urged us to verify whether a similar phenomenon could occur with tricyclo-DNA (tcDNA)-ASO that also display high-affinity properties to the target RNA. We thus ran a series of control experiments and demonstrate here that exon-skipping levels are not overestimated owing to an interference of tcDNA-ASO with the unskipped product in contrast to what was observed with LNA-containing ASO.

Small interfering RNAs (siRNAs) represent a novel class of drugs capable of potent and sustained modulation of genes across various tissues. Preclinical development of siRNAs necessitates assessing efficacy and toxicity in animal models. While identifying therapeutic leads with cross-species activity can expedite development, it may compromise efficacy and be infeasible for certain gene targets. Here, we investigate whether deriving species-active siRNAs from potent human-targeting leads-an approach termed mismatch conversion-can yield potent compounds. We systematically altered potent siRNAs targeting human genes associated with diseases-SOD1 (ALS), JAK1 (inflammation), and HTT (HD)-to generate species-matching variants with full complementarity to their target in NHPs, mice, rats, sheep, and dogs. Variants potency and efficacy were measured in corresponding cell lines. We demonstrate that sequence, position, and number of mismatches significantly influence the ability to generate potent species-active compounds via mismatch conversion. Across tested sequences, mismatch conversion strategy ability to identify a species-active lead varied from 0% to 70%. For SOD1, lead compounds identified from species-focus screening in mouse and dog cells were more potent than leads obtained from mismatch conversion. Thus, a focused screening of therapeutic lead and model compounds may represent a more reliable strategy for the clinical advancement of siRNAs.

Although CRISPR-Cas9 gene therapies have proven to be a powerful tool across many applications, improvements are necessary to increase the specificity of this technology. Cas9 cutting in off-target sites remains an issue that limits CRISPR's application in human-based therapies. Treatment of autosomal dominant diseases also remains a challenge when mutant alleles differ from the wild-type sequence by only one base pair. Here, we utilize synthetic peptide nucleic acids (PNAs) that bind selected spacer sequences in the guide RNA (gRNA) to increase Cas9 specificity up to 10-fold. We interrogate variations in PNA length, binding position, and degree of homology with the gRNA. Our findings reveal that PNAs bound in the region distal to the protospacer adjacent motif (PAM) site effectively enhance specificity in both on-target/off-target and allele-specific scenarios. In addition, we demonstrate that introducing deliberate mismatches between PNAs bound in the PAM-proximal region of the gRNA can modulate Cas9 activity in an allele-specific manner. These advancements hold promise for addressing current limitations and expanding the therapeutic potential of CRISPR technology.

Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5'-cytosine-phosphate-guanine-3' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.

Most oligonucleotide therapeutics use Watson-Crick-Franklin base-pairing hybridization to target RNA and mitigate disease-related protein production. Using targets that were previously inaccessible to small molecules and biologics, synthetic nucleotides have provided treatments for severely debilitating and life-threatening diseases. However, these therapeutics possess unique pharmacologies that require specific considerations for their distribution, clearance, and other clinical pharmacology characteristics. Namely, one hurdle in the drug development of these therapeutics remains the prediction of human dose that results in exposures comparable with or below those seen at no observed adverse effect level in animals. For first-in-human (FIH) clinical trials, this often involves allometric scaling based on body surface area (BSA) or body weight (BW). In this study, we reviewed the current literature and surveyed elements across 16 approved oligonucleotide therapeutic New Drug Applications approved by the U.S. Food and Drug Administration in the period from September 1998 to January 2024, and 89 Investigational New Drug (IND) programs with available FIH clinical trials conducted from January 2015 to January 2024, to understand dose selection in early-stage development of oligonucleotide therapeutics. The surveyed elements across these programs include study design, route of administration, dosing regimen, interspecies scaling approach, and the most sensitive species. Of 89 IND programs and 16 approved therapeutics, intravenous and subcutaneous were the most common route of administration, no observable adverse event levels were frequently derived from nonhuman primates, BSA and BW were adjusted for in similar frequencies, patients were predominantly enrolled in FIH trials, and the most common design was a single or multiple ascending dose trial.

Mass spectrometry (MS) has long been used for quality control of oligonucleotide therapeutics, including single-guide RNAs (sgRNAs) for clustered regularly interspaced short palindromic repeats techniques. However, the application of MS is limited to qualitative assays in most cases. Here, we showed that electrospray-ionization quadrupole time-of-flight MS (ESI-QTOF-MS) assays can be quantitative for chemical species found in sgRNA samples. More specifically, using a 100-nt SpCas9 sgRNA as the example, we estimated that the limits of quantification for length variants in the range of N - 4 to N + 4 (i.e., 96-104 nucleotides) were equal to or lower than 1%. Our study highlighted the potential of ESI-QTOF in its application as a quality control method for sgRNA molecules.