Nucleic acid therapeutics最新文献

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.

Although MYCN has been considered an undruggable target, MYCN alterations confer poor prognosis in many pediatric and adult cancers. The novel MYCN-specific inhibitor BGA002 is an antigene peptide nucleic acid oligonucleotide covalently bound to a nuclear localization signal peptide. In the present study, we characterized the pharmacokinetics (PK) of BGA002 after single and repeated administration to mice using a novel specific enzyme-linked immunosorbent assay. BGA002 concentrations in plasma showed linear PK, with dose proportional increase across the tested dose levels and similar exposure between male and female and between intravenous and subcutaneous route of administration. Repeated dosing resulted in no accumulation in plasma. Biodistribution up to 7 days after single subcutaneous administration of [¹⁴C]-radiolabeled BGA002 showed broad tissues and organ distribution (suggesting a potential capability to reach primary tumor and metastasis in several body sites), with high concentrations in kidney, liver, spleen, lymph nodes, adrenals, and bone marrow. Remarkably, we demonstrated that BGA002 concentrates in tumors after repeated systemic administrations in three mouse models with MYCN amplification (neuroblastoma, rhabdomyosarcoma, and small-cell lung cancer), leading to a significant reduction in tumor weight. Taking into account the available safety profile of BGA002, these data support further evaluation of BGA002 in patients with MYCN-positive tumors.

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene (HTT). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein in vivo and are considered a viable therapeutic strategy. However, the extent to which they silence huntingtin mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a divalent scaffold and delivered to two mouse models of HD. In both models, divalent siRNA sustained lowering of wild-type Htt, but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild-type HTT protein was observed in both models. Subsequent fluorescent in situ hybridization analysis shows that divalent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.