Biology subjects

Kennedy, J. G.

Publications and source records attributed to Kennedy, J. G..

3 recordsLinked to original sources

bioRxiv2025

Selective targeting of oncogenic KRAS G12D using peptide nucleic acid oligomers attached to cell-penetrating peptides

KRAS is a proto-oncogene that contains activating mutations in up to 30% of tumors. Many conventional therapies inhibit both cancerous and normal cells, which may cause toxicity. Thus, programmable mutant-selective targeted inhibitors are needed. Peptide nucleic acids (PNAs) incorporate base sequences analogous to DNA, with modified peptide backbones instead of ribose-phosphate backbones, allowing PNAs to hybridize with DNA with high avidity to suppress transcription. Here, we developed KRAS G12D-selective PNA oligomers with novel cell-penetrating flanking regions. Fluorescein-labeled PNA oligomers displayed high uptake rates in cells and nuclei. Exposure to PNA-delivery peptide conjugates resulted in repression of KRAS G12D mRNA and protein expression within 2 hours and lasting up to 48 hours. Varying cell-penetrating peptide (CPP) compositions and lengths of complementary KRAS sequences were tested using dose-response cell viability assays. These experiments identified configurations that were effective at selectively preventing growth of on-target KRAS G12D cells, while relatively sparing off-target KRAS G12C cells. Electrophoretic mobility shift assays demonstrated in vitro binding and selectivity for KRAS G12D DNA sequences. CPP-PNA-G12D-1 was effective against a panel of pancreatic ductal adenocarcinoma cell lines and patient-derived xenografts in vivo. These results show promise for an enhanced PNA-delivery peptide conjugate strategy as both a tool for studying tumors driven by oncogenic point mutations and as a potential therapeutic strategy to selectively target mutant cancer cells.

cancer biology↗

bioRxiv2025

Delayed lubricin injection improves cartilage repair tissue quality in an in vivo rabbit osteochondral defect model

Osteochondral lesions (OCL) are common among young patients and often require surgical interventions since cartilage has a poor capacity for self-repair. Bone marrow stimulation (BMS) has been used clinically for decades to treat OCLs, however a persisting challenge with BMS and other cartilage repair strategies is the inferior quality of the resulting fibrocartilaginous repair tissue. Lubrication-based therapies have the potential to improve the quality of cartilage repair tissue as joint lubrication is linked to local cartilage tissue strains and subsequent cellular responses including death and apoptosis. Recently, a full length recombinant human lubricin (rhLubricin) was developed and has been shown to lower friction in cartilage. This study investigated the effect of a single delayed injection of rhLubricin on cartilage repair in an in vivo rabbit OCL model using gross macroscopic evaluation, surface profilometry, histology, and tribology. Moderate improvement in macroscopic scores for cartilage repair were observed. Notably, quantitative analysis of Safranin-O histology showed that rhLubricin treated joints had significantly higher glycosaminoglycan content compared to saline treated joints, and there were no differences in repair integration between groups. Furthermore, rhLubricin treated joints had significantly lower friction coefficients tested across three sliding speeds compared to saline treated joints (rhLubricin: 0.15 {+/-} 0.03 at 0.1 mm/s to 0.12 {+/-} 0.03 at 10 mm/s, Saline: 0.22 {+/-} 0.06 at 0.1 mm/s to 0.19 {+/-} 0.05 at 10 mm/s). Overall, a single delayed injection of rhLubricin improved the quality and lubricating ability of the repair cartilage tissue without inhibiting repair tissue integration.

bioengineering↗

bioRxiv2024

Enhancing gRNA Transcript levels by Reducing the Scaffold Poly-T Tract for Optimal SpCas9- and SaCas9-mediated Gene Editing

Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols, the presence of these 4Ts did not significantly affect editing efficiency, as most of the gRNAs tested (55 gRNAs) achieved near-perfect editing outcomes. We observed that gRNAs with lower activity were T-rich and had reduced gRNA transcript levels. However, this issue can be effectively resolved by increasing transcript levels, which can be readily achieved by shortening the 4T sequences. In this study, we demonstrated this by modifying the sequences to 3TC. Although the 3TC scaffold modification did not improve editing efficiency for already efficient gRNAs when high vector quantities were available, it proved highly beneficial under conditions of limited vector availability, where the 3TC scaffold yielded higher editing efficiency. Additionally, we demonstrated that the 3TC scaffold is compatible with SpCas9 high-fidelity variants and ABEmax base editing, enhancing their editing efficiency. Another commonly used natural Cas9 variant, SaCas9, also benefited from the 3TC scaffold sequence modification, which increased gRNA transcription and subsequently improved editing activity. This modification was applied to the EDIT-101 therapeutic strategy, where it demonstrated marked improvements in performance. This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ensuring more effective and optimal outcomes.

molecular biology↗