Biology subjects

Lebedin, M.

Publications and source records attributed to Lebedin, M..

3 recordsLinked to original sources

bioRxiv2026

Efficient CRISPR/Cas9-mediated homology independent sequence replacement in vivo and non-dividing primary cells

Precise sequence replacement in non-dividing cells remains a major challenge for genome editing. Here we show that REPLACE (Rational end-joining protocol delivering a targeted sequence exchange), a homology-independent CRISPR/Cas9-based replacement strategy, enables exon- and gene-scale substitution in primary cells, in vivo tissues and post-mitotic human cardiomyocytes. REPLACE uses two guide RNAs to excise a defined genomic region and inserts a donor lacking homology arms through non-homologous end joining (NHEJ). In primary mouse hepatocytes, REPLACE mediated exon replacement in 35% of all cells. In adult mouse liver, editing efficiency could be increased to [~]20% when Cas9-sgRNA ribonucleoproteins were delivered via engineered virus-like particles (eVLPs) together with an adeno-associated virus (AAV) donor. REPLACE also supported large-segment replacement, enabling one-step exchange of a [~]27-kb mouse Ace2 interval with the human ACE2 coding region in zygotes, followed by germline transmission and tissue-specific expression. Finally, we applied REPLACE to a disease-relevant mutation that is not readily addressable by base editing and was poorly corrected by prime editing in post-mitotic cardiomyocytes. At the LMNA locus, REPLACE corrected the K117fs frameshift mutation in patient-derived post-mitotic cardiomyocytes with precise exon replacement and restored Lamin A/C protein expression and nuclear lamina localization. These findings establish REPLACE as a versatile platform for homology-independent sequence replacement and as a complementary approach for genetic correction in settings where homology-directed repair (HDR), base editing (BE) or prime editing (PE) are inefficient or not applicable.

genomics↗

bioRxiv2024

SARS-CoV-2 neutralization and protection of hamsters via nasal administration of a humanized neutralizing antibody

Monoclonal antibodies are widely used for the treatment of infectious human diseases, including COVID-19. Since the start of the pandemic, eight monoclonal antibodies against SARS-CoV-2 were granted emergency use authorization. High mutation rate of the SARS-CoV-2 virus has led to the emergence of highly transmissible variants efficiently evading vaccine-induced immunity. This highlights the importance of identifying broadly neutralizing antibodies with therapeutic potential. In this study, we used a panel of murine monoclonal antibodies (mAb) to identify a subset that bound and neutralized a broad spectrum of SARS-CoV-2 variants. Intranasal delivery of XR10, the most promising murine mAb, protected hamsters against infection by Alpha and Delta variants. We next humanized XR10 mAb using a combination of CDR-grafting and Vernier zones preservation approaches (CRVZ) to create a panel of humanized antibody variants. We ranked the variants based on their spike binding ability and virus neutralization. Of these, XR10v48 demonstrated the best ability to neutralize SARS-CoV-2 variants. XR10v48 was protective in hamsters when given as a single 50 {micro}g/kg intranasal dose at the time of viral challenge. XR10v48 features 34 key amino acid residues retained from the murine progenitor. Our work introduces a potent humanized antibody that demonstrates neutralizing activity in vivo at a low dose.

immunology↗

bioRxiv2020

A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

Precision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method Replace editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

genetics↗