Publications

he CRISPR gene editing tool holds great potential for curing genetic disorders. However, the safe, efficient, and specific delivery of the CRISPR/Cas9 components into cells and tissues remains a challenge. While many currently available delivery methods achieve high levels of gene editing effects in vivo, they often result in genotoxicity and immunogenicity. Extracellular vesicles (EVs), which are cell-derived lipid nanoparticles, are capable of transferring protein and nucleic acid cargoes between cells, making them a promising endogenous alternative to synthetic delivery methods. This review provides a comprehensive analysis of the currently available strategies for EV-mediated delivery of CRISPR/Cas9. These strategies include cell-based, passive loading obtained by overexpression of CRISPR/Cas9, active loading involving protein or RNA dimerization, and loading into already purified EVs. All these approaches suggest that EV-based CRISPR/Cas9 delivery is useful for achieving both in vitro and in vivo gene editing. Despite that, substantial variations in cellular uptake and gene editing efficiencies indicate that further improvement and standardization are required for the therapeutic use of EVs as a CRISPR/Cas9 delivery vehicle. These improvements include, but is not limited to, the high-yield purification of EVs, increased loading and release efficiencies, as well as improved tissue- or cell-specific targeting specificities.

Human enhancement involves interventions that enhance physical or cognitive performance or overall wellbeing beyond typical human limits. The consequence of human enhancement has the potential to radically reshape society and the very existence of our species, requiring careful consideration of its ethical and regulatory boundaries. In this paper, we explore current applications of human enhancement, including motor neuro -prostheses, transcranial magnetic stimulation, and gene therapy. We analysed key unresolved challenges and potential solutions, focusing on regulatory and policy frameworks, ethical considerations in clinical research, transparency through registries, traceability of interventions, and the role of informed decision-making and consent. Finally, we critically examine the evolving definition of health and the need to delineate clear boundaries between prevention and enhancement. By addressing these issues, this paper aims to contribute to the ongoing dialog on the responsible development and implementation of human enhancement technologies.

β-Thalassemia is brought about by defective β-globin (HBB [hemoglobin subunit β]) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break-based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBB(IVSI-110(G>A)) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34(+) cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBB(IVSI-110(G>A)) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.

Targeted nucleases, primarily CRISPR-Cas-based systems, have revolutionized genome editing by enabling precise modification of target genes or transcripts. Many pre-clinical and clinical studies leverage this technology to develop treatments for human diseases; however, substantial off-target genotoxicity concerns delay its clinical translation. Despite the development of a wide array of tools, assays, and technologies aimed at identifying and quantifying off-target effects, the absence of standardized guidelines leads to inconsistent
practices across studies. This review highlights the key challenges and potential solutions in ensuring the safety of gene editing studies for therapeutic applications, focusing on gRNA design, off-target sites prediction, and off-target activity measurement.