Wave’s PRISM platform enables the generation of chimeric backbone-containing stereopure oligonucleotides with improved pharmacological properties through position-controlled chemistry and stereochemical configuration. Here, we will provide an update on our progress in developing oligonucleotides that are optimized for distinct high-priority genetic targets, modalities, and tissues. As illustration, we will describe our development of stereopure phosphoryl guanidine (PN) backbone-containing chimeric oligonucleotides for RNA interference (RNAi) applications in hepatic and extrahepatic tissues.  

siRNAs have the potential to target many genetically defined disorders. In this study we investigate the impact of chemical architecture and route of administration on siRNA delivery to human skin ex vivo and pig skin in vivo. Our data outline an effective strategy for functional delivery of siRNAs enabling efficient silencing of genetic targets for dermal indications.

Most refractory malignant solid tumors create an immunosuppressive tumor microenvironment (TME), rendering them resistant to standard-of-care immune checkpoint inhibitors (CPIs). We investigated the feasibility of applying systemically delivered RNAi agent to silence the mRNA of a historically undruggable target, that involved in mediating immune suppression in TME, directly in tumor-associated immune cells. In preclinical rodent tumor models, GalXC-Plus conjugates selectively silenced the mRNA in multiple relevant cell types, reduced the protein levels, and increased cytotoxic T-cell infiltration. In immunotherapy resistant tumors, RNAi-mediated silencing resulted in tumor growth inhibition, which was further enhanced in combination with CPIs. A human active STAT3 conjugate is currently in Phase 1 clinical trial for treatment of immunotherapy refractory cancer types (NCT06098651).

We developed an unconventional GalNAc-based RNAi therapeutic technology, GalAhead, comprising two key components – mxRNA (miniaturized RNAi triggers) and muRNA (multi-unit RNAi triggers). mxRNAs are composed of single ~30nt oligonucleotides and downregulate individual genes, while muRNAs are comprised of 2 or more oligonucleotides and can simultaneously silence two or more targets. We will present data demonstrating validity of the GalAhead technology in vitro and in vivo, as well as progress report with quickly expanding and progressing GalAhead-based therapeutic pipeline, with first program entering clinical trials earlier this year.

Nonclinical safety screening of GalNAc-siRNAs is typically carried out in rats at exaggerated exposures in a repeat-dose regimen. We have previously shown that at these suprapharmacological doses, hepatotoxicity observed with a subset of GalNAc-siRNAs is driven by antisense strand seed-mediated off-target activity. To increase specificity, we developed a novel design strategy termed ESC+, which utilizes chemical modifications that thermally destabilize the base pairing between the seed region and off-target mRNAs.

Antibody-oligonucleotide conjugate (AOC) technology is a promising approach to facilitate the functional delivery of oligonucleotides to a variety of target tissues. We utilized targeting TfR1 to deliver therapeutic oligonucleotides to muscle tissue for the treatment of rare neuromuscular diseases. The preclinical systemic and tissue pharmacokinetics of AOCs in vivo will be discussed, including strategies to maximize receptor-mediated tissue uptake.

Vertebrate immune systems have evolved nucleic acid immune sensing receptors to detect the presence of foreign nucleic acids, and to activate innate and adaptive immune defenses. Synthetic RNA and DNA ligands for these receptors induce antibody and T cell responses that underly the efficacy of mRNA vaccines and may be used for in situ immunization to treat cancer. Understanding the different biology of these receptors, including TLR3, TLR7, TLR8, TLR9, cGAS/STING, and the RIG-I-like receptors is essential for optimizing cancer immunotherapy in humans.

We developed a new treatment strategy by modifying tumor suppressor miRNAs, hsa-miRNA-15a (miR-15a) with gemcitabine (Gem), to create Gem-modified mimics of miR-15a (Gem-miR-15a). Gem-miR-15a is a potent inhibitor to eliminate patient-derived pancreatic cancer organoids and mouse tumor models without delivery vehicle.

To overcome current limitations of oligonucleotide therapeutic delivery, we have designed a family of proprietary cyclic CPPs that form the core of our Endosomal Escape Vehicle (EEV) technology and covalently conjugated it to oligonucleotides. Using preclinical models of Duchenne muscular dystrophy (DMD), we demonstrated the ability of our EEV platform technology to efficiently deliver oligonucleotides to skeletal and cardiac muscle, the primary sites of pathology in DMD.

DelSiTech technology offers a way to deliver APIs over an extended period by encapsulating them in biodegradable silica. Our recent findings on injectable, long-acting antisense oligonucleotides demonstrate the capability of the technology to support the generation of durable treatments, from sensitive and often highly complex molecules. Join us as we explore our technology and how it can support the progression of long-lasting nucleic acid–based therapies such as oligonucleotide, mRNA, and more.

• What are the current chemistry demands and promising strategies towards successful therapeutic oligonucleotides for extrahepatic tissues?
-Oligonucleotide backbone modification
-Delivery strategies
-Strategies to modulate cell-type specificity
-Strategies to facilitate endosomal escape

• How can we further amplify the benefits of collaboration between academic labs and biotechs?
  -Ideal research partnerships between academic labs and biotechs that synergistically facilitate oligonucleotide drug development
  -Future path for n=1 therapeutics

Developments from Early Discovery Through to Late-Stage Clinical Programs

• How do you know your discovery should move toward the clinic
• Patents
• Steps toward the clinic-biology/biochemistry/chemistry, cell biology, animal studies, toxicology, and who/how to carry out a clinical trial

Strategic Collaboration Between Big Pharma and Small Biotechs

• How do you define a strategic collaboration-from the perspective of big pharma and small biotech (i.e. why a collaboration)
• Conditions for a strategic/successful collaboration
• Oligonucleotide CMC and Regulatory Strategies​

The immunosuppressive tumor microenvironment is an efficient barrier that protects the tumor from destruction by the immune system. It comprises several targets that are predestined as targets for therapeutic antisense oligonucleotides (ASOs). As an example, we will present data showing that ASOs suppressing expression of the multifactorial protein NRP1 are a promising option for treatment of cancer.

Functionalization of therapeutic oligonucleotides by conjugating to multiple modalities may be beneficial for their efficacy or delivery. Certain examples like GalNAc have been widely applied in therapeutic ASO and siRNA development. Maleimide-conjugated ASO can be easily functionalized by conjugating to thiol-bearing modalities. During the manufacturing development of such conjugates we have encountered several difficulties, conquered them, and learned lessons. This presentation intends to share what we learned.

This presentation introduces a novel nanoarchitecture, BROTHERS or BRO, designed to mitigate off-target interactions of antisense oligonucleotides (ASOs). The BRO, comprising a typical gapmer ASO and a complementary peptide nucleic acid (PNA) strand, deters non-specific protein and RNA binding. Harnessing its inherent free energy, BRO triggers a toehold-mediated strand displacement reaction, which enhances ASO's mismatch recognition, minimizing hybridization to RNA off-targets. Consequently, BRO enhances therapeutic window of ASOs.