Preclinical and clinical developments in enzyme-loaded red blood cells: an update.

INTRODUCTION: We have previously described the preclinical developments in enzyme-loaded red blood cells to be used in the treatment of several rare diseases, as well as in chronic conditions. AREA COVERED: Since our previous publication we have seen further progress in the previously discussed approaches and, interestingly enough, in additional new studies that further strengthen the idea that red blood cell-based therapeutics may have unique advantages over conventional enzyme replacement therapies in terms of efficacy and safety. Here we highlight these investigations and compare, when possible, the reported results versus the current therapeutic approaches. EXPERT OPINION: The continuous increase in the number of new potential applications and the progress from the encapsulation of a single enzyme to the engineering of an entire metabolic pathway open the field to unexpected developments and confirm the role of red blood cells as cellular bioreactors that can be conveniently manipulated to acquire useful therapeutic metabolic abilities. Positioning of these new approaches versus newly approved drugs is essential for the successful transition of this technology from the preclinical to the clinical stage and hopefully to final approval.

Development and in vitro characterization of a humanized scFv against fungal infections.

The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against ß-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind ß-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs.

The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker ß-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.

A negative feedback mechanism links UBC gene expression to ubiquitin levels by affecting RNA splicing rather than transcription.

UBC gene plays a critical role in maintaining ubiquitin (Ub) homeostasis. It is upregulated under stress conditions, and herein we report that it is downregulated upon Ub overexpression. Downregulation occurs in a dose-dependent manner, suggesting the existence of a fine-tuned Ub sensing mechanism. This "sensor" requires a conjugation competent ubiquitin to detect Ub levels. Searching the sensor among the transcription factors involved in basal and stress-induced UBC gene expression was unsuccessful. Neither HSF1 and HSF2, nor Sp1 and YY1 are affected by the increased Ub levels. Moreover, mutagenesis of their binding sites in the UBC promoter-driven reporter constructs does not impair the downmodulation effect. Epigenetic studies show that H2A and H2B ubiquitination within the UBC promoter region is unchanged upon ubiquitin overexpression. Noteworthy, quantification of nascent RNA molecules excludes that the downmodulation arises in the transcription initiation step, rather pointing towards a post-transcriptional mechanism. Indeed, a significantly higher fraction of unspliced UBC mRNA is detected in ubiquitin overexpressing cells, compared to empty vector transfected cells. Our findings suggest how increasing cellular ubiquitin levels may control the expression of UBC gene by negatively affecting the splicing of its pre-mRNA, providing a straightforward feedback strategy for the homeostatic control of ubiquitin pools.