Fernando Pastor

Viroimmunotherapy leverages oncolytic viruses to induce antitumor immunity and is increasingly explored for solid tumors. Their activity can be enhanced by arming them with immunostimulatory payloads, but most approaches rely on protein-based transgenes that are constrained by viral genome packaging limits. Here, we establish a replication-competent Delta-24-RGD-based platform for localized production of immunotherapeutic RNA aptamers at the tumor site. RNA aptamers provide compact, highly specific ligands that can, in principle, target diverse immune receptors. As a model, we engineered a Delta-24-RGD derivative encoding circular 4-1BB targeting aptamers and show that infected tumor cells sustain aptamer transcription and release, which is associated with a pro-inflammatory remodeling of the tumor microenvironment and measurable antitumor activity in different mouse models with a comparable effect to that achieved with a 4-1BBL-expressing adenovirus used as a benchmark. Overall, this work delivers a proof of concept that replication-competent adenoviruses can serve as in situ factories for extracellularly active RNA aptamers, supporting their development as flexible platforms for localized non-coding cancer immunotherapy.

The ATP-hydrolytic ectoenzyme ENPP1 has been implicated in the metastasis and recurrence in triple-negative breast cancer (TNBC), primarily by contributing to tumor cell survival and treatment resistance. However, the precise mechanisms remain unclear. In a model of local recurrence (LR), circulating tumor cells (CTC) engrafting in the post-resection tumor bed developed a radioresistant phenotype linked to an ENPP1⁺-gene signature which was also identified in TNBC patients, suggesting ENPP1´s role in genome integrity. Blockade of ENPP1 using a permeable ENPP1 inhibitor (AVA-NP-695) reduced radioresistance, mechanistically attributed to decreased homologous recombination (HR) resulting in persistent DNA damage, as evidenced by enhanced tail moment and sustained γH2AX formation. This impaired DNA damage repair (DDR) sensitized tumor cells to ionizing radiation (IR). Notably, several DDR inhibitors (i) (including PARPi and ATMi) showed the highest synergy score in a targeted pharmacological screening. In vivo, dual ENPP1/ATM inhibition heightened radiosensitivity, compromised tumor cell survival and enhanced STING-TBK1 signaling by preventing ENPP1-mediated cGAMP hydrolysis. This resulted in robust innate and long-lasting adaptive antitumor immune memory responses, leading to significant tumor regression. Remarkably, combined treatment post-IR reduced spontaneous metastasis and local recurrence, and induced abscopal effects that impacted distant tumor spread in orthotopic tumor models. Thus, these findings position ENPP1 as a critical link between genome integrity and immunosuppression, offering promising translational opportunities for treating local or distant dissemination in TNBC.

RNA-based therapeutics have rapidly emerged over the past decade, offering a new class of medicines that differ significantly from conventional drugs. These therapies can be programmed to target or restore defective genes, allowing for more personalised treatments and reducing side effects. Notably, RNA therapies have made significant progress in the treatment of genetic liver diseases, exemplified by small interfering RNA treatments for hereditary transthyretin amyloidosis, which use liver-targeting strategies such as GalNAc conjugation to improve efficacy and safety. RNA-based gene-editing technologies, such as base editor and prime editor clustered regularly interspaced short palindromic repeats systems, also show promise with their ability to minimise genomic rearrangements and cancer risk. While RNA therapies offer high precision, challenges remain in optimising delivery methods and ensuring long-term safety and efficacy. Lipid nanoparticle-mRNA therapeutics, particularly for protein replacement in rare diseases, have gained support from preclinical successes. Compared with viral gene therapies, mRNA therapies present a safer profile with reduced risks of genomic integration and oncogene activation. However, clinical trials, especially for rare diseases, face limitations such as small sample sizes and short observation periods. Further preclinical studies, including non-human primates, will be essential for refining trial designs. Despite their potential, the high costs of RNA therapies pose a challenge that will require cost–utility models to guide pricing and accessibility. Here, we discuss the fundamental aspects of RNA-based therapeutics and showcase the most relevant preclinical and clinical developments in genetic liver metabolic diseases.

Pediatric diffuse midline gliomas (DMG) with altered H3-K27M are aggressive brain tumors that arise during childhood. Despite advances in genomic knowledge and the significant number of clinical trials testing new targeted therapies, patient outcomes are still poor. Immune checkpoint blockades with small molecules, such as aptamers, are opening new therapeutic options that represent hope for this orphan disease. Here, we demonstrated that a TIM-3 aptamer (TIM-3 Apt) as monotherapy increased the immune infiltration and elicited a strong specific immune response with a tendency to improve the overall survival of treated DMG-bearing mice. Importantly, combining TIM-3 Apt with radiotherapy increased the overall median survival and led to long-term survivor mice in 2 pediatric DMG orthotopic murine models. Interestingly, TIM-3 Apt administration increased the number of myeloid populations and the proinflammatory CD8-to-Tregs ratios in the tumor microenvironment as compared with nontreated groups after radiotherapy. Importantly, the depletion of T cells led to a major loss of the therapeutic effect achieved by the combination. This work uncovers TIM-3 targeting as an immunotherapy approach to improve the radiotherapy outcome in DMGs and offers a strong foundation for propelling a phase I clinical trial using radiotherapy and TIM-3 blockade combination as a treatment for these tumors.