RNA editing: Expanding the potential of RNA therapeutics.

RNA therapeutics have had a tremendous impact on medicine, recently exemplified by the rapid development and deployment of mRNA vaccines to combat the COVID-19 pandemic. In addition, RNA-targeting drugs have been developed for diseases with significant unmet medical needs through selective mRNA knockdown or modulation of pre-mRNA splicing. Recently, RNA editing, particularly antisense RNA-guided adenosine deaminase acting on RNA (ADAR)-based programmable A-to-I editing, has emerged as a powerful tool to manipulate RNA to enable correction of disease-causing mutations and modulate gene expression and protein function. Beyond correcting pathogenic mutations, the technology is particularly well suited for therapeutic applications that require a transient pharmacodynamic effect, such as the treatment of acute pain, obesity, viral infection, and inflammation, where it would be undesirable to introduce permanent alterations to the genome. Furthermore, transient modulation of protein function, such as altering the active sites of enzymes or the interface of protein-protein interactions, opens the door to therapeutic avenues ranging from regenerative medicine to oncology. These emerging RNA-editing-based toolsets are poised to broadly impact biotechnology and therapeutic applications. Here, we review the emerging field of therapeutic RNA editing, highlight recent laboratory advancements, and discuss the key challenges on the path to clinical development.

GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell-derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

Modeling Therapeutic Antibody-Small Molecule Drug-Drug Interactions Using a Three-Dimensional Perfusable Human Liver Coculture Platform.

Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced three-dimensional (3D) liver coculture platforms offer the potential for extended hepatocyte functionality and allow for the study of more complex biologic interactions, which can improve and refine human drug safety evaluations. Here, we use a perfusion flow 3D microreactor platform for the coculture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of cytochrome P450 3A4 isoform (CYP3A4) activity by chronic interleukin 6 (IL-6)-mediated inflammation over 2 weeks. Hepatocyte cultures remained stable over 2 weeks, with consistent albumin production and basal IL-6 levels. Direct IL-6 stimulation that mimics an inflammatory state induced a dose-dependent suppression of CYP3A4 activity, an increase in C-reactive protein (CRP) secretion, and a decrease in shed soluble interleukin-6 receptor (IL-6R) levels, indicating expected hepatic IL-6 bioactivity. Tocilizumab, an anti-IL-6R monoclonal antibody used to treat rheumatoid arthritis, has been demonstrated clinically to impact small molecule drug pharmacokinetics by modulating cytochrome P450 enzyme activities, an effect not observed in traditional hepatic cultures. We have now recapitulated the clinical observation in a 3D bioreactor system. Tocilizumab was shown to desuppress CYP3A4 activity while reducing the CRP concentration after 72 hours in the continued presence of IL-6. This change in CYP3A4 activity decreased the half-life and area under the curve up to the last measurable concentration (AUClast) of the small molecule CYP3A4 substrate simvastatin hydroxy acid, measured before and after tocilizumab treatment. We conclude that next-generation in vitro liver culture platforms are well suited for these types of long-term treatment studies and show promise for improved drug safety assessment.

A tough, precision-porous hydrogel scaffold: ophthalmologic applications.

Appropriate mechanical properties and highly interconnected porosity are important properties for tissue engineering scaffolds. However, most existing hydrogel scaffolds suffer from poor mechanical properties limiting their application. Furthermore, it is relatively infrequent that precision control is achieved over pore size and structure of the scaffold because there are relatively few current technologies that allow such control and there is not a general appreciation that such control is important. To address these shortcomings, by combining double network polymerization and sphere-templating fabrication techniques, we developed a tough, intelligent scaffold based on poly(acrylic acid) and poly(N-isopropyl acrylamide) with a controllable, uniform, and interconnected porous structure. A mechanical assessment showed the toughness of the hydrogel and scaffold to be up to ∼1.4 × 10(7) Jm(-3) and ∼1.5 × 10(6) Jm(-3) respectively, as compared with 10(4)-10(5) Jm(-3) for most synthetic hydrogels. The thermosensitivity and pH-sensitivity were explored in a swelling study. In vitro testing demonstrated the scaffold matrices supported NIH-3T3 cell adhesion, proliferation and infiltration. An in vivo rabbit study showed the scaffolds promote strong cellular integration by allowing cells to migrate into the porous structure from the surrounding tissues. These data suggest that the poly(acrylic acid)/poly(N-isopropyl acrylamide)-based scaffold could be an attractive candidate for tissue engineering.

Prostate cancer xenografts engineered from 3D precision-porous poly(2-hydroxyethyl methacrylate) hydrogels as models for tumorigenesis and dormancy escape.

Synthetic biomaterial scaffolds show promise for in vitro and in vivo 3D cancer models. Tumors engineered in biomaterial scaffolds have shown evidence of being more physiologically relevant than some traditional preclinical model systems, and synthetic biomaterials provide the added benefit of defined and consistent microenvironmental control. Here, we examine sphere-templated poly(2-hydroxyethyl methacrylate) (pHEMA) scaffolds as the basis for engineering xenografts from multiple human prostate cancer cell lines. pHEMA scaffolds seeded and pre-cultured with tumorigenic M12 cells prior to implantation generated tumors in athymic nude mice, demonstrating the ability of the scaffolds to be used as a synthetic vehicle for xenograft generation. pHEMA scaffolds seeded with LNCaP C4-2 cells, which require Matrigel or stromal cell support for tumor formation, were poorly tumorigenic up to 12 weeks after implantation even when Matrigel was infused into the scaffold, demonstrating a lack of necessary pro-tumorigenic signaling within the scaffolds. Finally, M12mac25 cells, which are ordinarily rendered non-tumorigenic through the expression of the tumor suppressor insulin-like growth factor binding protein 7 (IGFBP7), displayed a tumorigenic response when implanted within porous pHEMA scaffolds. These M12mac25 tumors showed significant macrophage infiltration within the scaffolds driven by the foreign body response to the materials. These findings show the potential for this biomaterials-based model system to be used in the study of prostate cancer tumorigenesis and dormancy escape.

Capillary force seeding of sphere-templated hydrogels for tissue-engineered prostate cancer xenografts.

Biomaterial-based tissue-engineered tumor models are now widely used in cancer biology studies. However, specific methods for efficient and reliable cell seeding into these and tissue-engineering constructs used for regenerative medicine often remain poorly defined. Here, we describe a capillary force-based method for seeding the human prostate cancer cell lines M12 and LNCaP C4-2 into sphere-templated poly(2-hydroxyethyl methacrylate) hydrogels. The capillary force seeding method improved the cell number and distribution within the porous scaffolds compared to well-established protocols such as static and centrifugation seeding. Seeding efficiency was found to be strongly dependent on the rounded cell diameter relative to the pore diameter and pore interconnect size, parameters that can be controllably modulated during scaffold fabrication. Cell seeding efficiency was evaluated quantitatively using a PicoGreen DNA assay, which demonstrated some variation in cell retention using the capillary force method. When cultured within the porous hydrogels, both cell lines attached and proliferated within the network, but histology showed the formation of a necrotic zone by 7 days likely due to oxygen and nutrient diffusional limitations. The necrotic zone thickness was decreased by dynamically culturing cells in an orbital shaker. Proliferation analysis showed that despite a variable seeding efficiency, by 7 days in culture, scaffolds contained a roughly consistent number of cells as they proliferated to fill the pores of the scaffold. These studies demonstrate that sphere-templated polymeric scaffolds have the potential to serve as an adaptable cell culture substrate for engineering a three-dimensional prostate cancer model.