Elias Quijano

There is intense interest in the advancement of RNAs as rationally designed therapeutic agents, especially in oncology, where a major focus is to use RNAs to stimulate pattern recognition receptors to leverage innate immune responses. However, the inability to selectively deliver therapeutic RNAs within target cells after intravenous administration now hinders the development of this type of treatment for cancer and other disorders. Here, we found that a tumor-targeting, cell-penetrating, and RNA binding monoclonal antibody, TMAB3, can form stable, noncovalent antibody/RNA complexes of a discrete size that mediate highly specific and functional delivery of RNAs into tumors. Using 3p-hpRNA, an agonist of the pattern recognition receptor retinoic acid–inducible gene-I (RIG-I), we observed robust antitumor efficacy of systemically administered TMAB3/3p-hpRNA complexes in mouse models of pancreatic cancer, medulloblastoma, and melanoma. In the KPC syngeneic, orthotopic pancreatic cancer model in immunocompetent mice, treatment with TMAB3/3p-hpRNA tripled animal survival, decreased tumor growth, and specifically targeted malignant cells, with a 1500-fold difference in RNA delivery into tumor cells versus nonmalignant cells within the tumor mass. Single-cell RNA sequencing (scRNA-seq) and flow cytometry demonstrated that TMAB3/3p-hpRNA treatment elicited a potent antitumoral immune response characterized by RIG-I activation and increased infiltration and activity of cytotoxic T cells. These studies established that TMAB3/RNA complexes can deliver RNA payloads specifically to hard-to-treat tumor cells to achieve antitumor efficacy, providing an antibody-based platform to advance the study of RNA therapies for the treatment of patients with cancer.

In utero gene editing has the potential to modify disease causing genes in multiple developing tissues before birth, possibly allowing for normal organ development, disease improvement, and conceivably, cure. In cystic fibrosis (CF), a disease that arises from mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, there are signs of multiorgan disease affecting the function of the respiratory, gastrointestinal, and reproductive systems already present at birth. Thus, treating CF patients early is crucial for preventing or delaying irreversible organ damage. Here we demonstrate proof-of-concept of multiorgan mutation correction in CF using peptide nucleic acids (PNAs) encapsulated in polymeric nanoparticles and delivered systemically in utero. In utero editing was associated with sustained postnatal CFTR activity, at a level similar to that of wild-type mice, in both respiratory and gastrointestinal tissue, without detection of off-target mutations in partially homologous loci. This work suggests that systemic in utero gene editing represents a viable strategy for treating monogenic diseases before birth that impact multiple tissue types.

Through preimplantation genetic diagnosis, genetic diseases can be detected during the early stages of embryogenesis, but effective treatments for many of these disorders are lacking. Gene editing could allow for correction of the underlying mutation during embryogenesis to prevent disease pathogenesis or even provide a cure. Here, we demonstrate that administration of peptide nucleic acids and single‐stranded donor DNA oligonucleotides encapsulated in poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles to single‐cell embryos allows for editing of an eGFP‐beta globin fusion transgene. Blastocysts from treated embryos exhibit high levels of editing (~94%), normal physiological development, normal morphology, and no detected off‐target genomic effects. Treated embryos reimplanted to surrogate moms show normal growth without gross developmental abnormalities and with no identified off‐target effects. Mice from reimplanted embryos consistently show editing, characterized by mosaicism across multiple organs with some organ biopsies showing up to 100% editing. This proof‐of‐concept work demonstrates for the first time the use of peptide nucleic acid (PNA)/DNA nanoparticles as a means to achieve embryonic gene editing.

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.

There is intense interest in the development of nucleic acid ligands for immune stimulation of the tumor microenvironment via pattern recognition receptors, especially for the treatment of “cold” tumors. However, delivery of these ligands in a tumor-specific manner to avoid systemic toxicity has been a challenge. Many current efforts rely on direct intra-tumoral injection of RNAs or other stimulatory immune ligands, which is therapeutically sub-optimal, especially for metastatic disease. Here we report studies evaluating the utility of a lupus-derived, cell-penetrating antibody, to deliver nucleic acids including mRNA to tumors in vivo. This antibody, a modified version of 3E10-D31N, forms non-covalent complexes with RNAs and can mediate highly specific delivery broadly into tumors via intravenous injection by targeting the nucleoside transporter ENT2. Studies with labeled RNAs and with mRNAs expressing a GFP reporter gene show tumor specific delivery and functional expression by imaging, with minimal delivery to healthy tissues. Cell culture studies show antibody-mediated delivery of a series of RIG-I ligands, including hairpin RNAs and poly(I:C), resulting in robust RIG-I stimulation and induction of type-1 interferon signaling. Using mouse B16 melanoma tumors as a model, we observe substantial tumor growth suppression upon i.v. injection of a combination of 3E10 complexed with poly(I:C) (synthetic double-stranded RNA). These results highlight a novel approach for the systemic delivery of immunostimulatory RNAs in a targeted manner that may offer treatment advantages over current approaches. Citation Format: Elias Quijano, Yanfeng Liu, Stephen Squinto, Bruce C. Turner, Peter M. Glazer. Systemic Administration of an antibody/RNA complex results in tumor specific delivery of immunostimulatory RNAs and tumor growth suppression in a mouse model of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB169.

Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of β-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.

We appreciate Pandey et al.’s (1) interest in further understanding our published work (2). Our responses are given below the excerpts from the Letter.Our first and foremost concern is the data presented in their figure 6C. We fail to understand why there was a fall in the plasma viral loads after day 19 in the control group (i.e., the group not given any intervention). Furthermore, not only was the fall in the plasma viral load significant (to below the limit of detection; <150 copies per milliliter) but also it was more rapid (showing a steeper decline) compared with the treated groups by day 32. If the viral loads are decreasing so rapidly and significantly in the noninterventional (control group), can we draw conclusions about the efficacy of … [↵][1]1To whom correspondence should be addressed. Email: william.jorgensen{at}yale.edu. [1]: #xref-corresp-1-1