Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.

Peptides from heptad repeat (HR1 and HR2) regions of gp41 are effective inhibitors of HIV-1 entry that block the fusion of viral and cellular membranes, but the generation of antibodies highly specific for these peptides is challenging. We have previously described a mouse hybridoma that recognizes MT-C34-related peptides derived from HR2. It was used for the selection of HIV-1-resistant CD4 lymphocytes engineered to express the MT-C34 peptide via a CRISPR/Cas9-mediated knock-in into the CXCR4 locus. In this study, we cloned variable domains of this antibody and generated a recombinant chimeric antibody (chAb) by combining it with the constant regions of the humanized antibody Trastuzumab. The new chAb displayed a high specificity and two-fold higher level of affinity than the parental mouse monoclonal antibody. In addition, chAb mediated up to 27–43% of the antibody-dependent cellular cytotoxicity towards cells expressing MT-C34 on their surface. The anti-MT-C34 chAb can be easily generated using plasmids available for the research community and can serve as a valuable tool for the detection, purification, and even subsequent elimination of HIV-1-resistant CD4 cells or CAR cells engineered to fight HIV-1 infection.

Radiotherapy (RT) remains a cornerstone in breast cancer (BC) treatment. While RT is effective, exposure to surrounding healthy tissues can lead to chromosomal damage and potentially increase the risk of secondary cancers. This study investigated chromosomal damage in circulating lymphocytes of BC patients receiving different RT schemes: ultra-hypofractionation in five fractions (UHF) and moderate hypofractionation in either 10-15 fractions (MHF-low) or 20 fractions (MHF-high). We also aimed to investigate how RT parameters influence MN yields in lymphocytes of BC patients. Additionally, the in vitro chromosomal radiosensitivity of primary and second primary BC patients was evaluated. Blood samples were collected pre- and post-RT from 182 primary and 50 second primary BC patients. Chromosomal damage was assessed in lymphocytes using the cytokinesis-block micronucleus (CBMN) assay. To evaluate chromosomal radiosensitivity, all blood samples were irradiated in vitro with 1 Gy of X-ray before performing the CBMN assay. Associations of MN yields with cancer type, RT scheme, lymph node irradiation, boost dose delivery, and irradiated body volume were analyzed using linear regression and linear mixed-effects models. Our findings indicated no difference in MN yields between primary and second primary BC patients. MHF-high RT scheme was associated with the highest MN induction, while the UHF scheme resulted in the lowest. Additionally, increased chromosomal damage was correlated with higher total tumor doses, larger irradiated volumes, and lymph node irradiation. No significant difference in in vitro chromosomal radiosensitivity was observed between primary and second primary BC patients. Shorter RT scheme with higher dose per fraction may mitigate cytogenetic damage in circulating lymphocytes, offering potential advantages in long-term safety profiles. In vitro chromosomal radiosensitivity, as assessed by CBMN assay is not a hallmark for second primary BC risk.

Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized the study of gene and genome function and genetic disease correction. While numerous genetically modified cellular and animal models have been created to understand biological processes, the clinical application of CRISPR/Cas tools has been impeded by off-targeting and delivery problems. It is generally accepted that the delivery of CRISPR in the form of a ribonucleoprotein complex (RNP) substantially reduces the time of DNA exposure to the effector nuclease, minimizing off-target effects and facilitating clinical usage. This review focuses on CRISPR/Cas RNP delivery with retro/lentiviral particles and exosomes, whose parallel production by cells transfected with viral vectors is underestimated. We critically evaluate specific mechanisms of extracellular particle formation and loading with CRISPR/Cas for each system. Additionally, the details of Cas-nanoparticle entry and uncoating, previously unappreciated in the context of gene editing efficiency, are discussed. Based on existing knowledge about the consequences of intervention in retroviral assembly, entry, or exosome formation, we outline the potential problems with CRISPR/Cas delivery using extracellular nanoparticles and ways to address them.