Gene therapy, a revolutionary approach promising to prevent and cure diseases by manipulating gene expression within a patient’s cells, faces a significant hurdle: ensuring the new gene reaches the cell’s nucleus. This challenge has impeded the advancement of treatments. However, researchers at the University of California San Diego, led by Professor Neal Devaraj from the Department of Biochemistry and Molecular Biophysics, have developed a novel method that significantly improves gene delivery efficiency while minimizing harmful side effects. Their findings were published in the prestigious journal Nature Communications.
For gene therapeutics to be effective, the introduced gene must successfully travel to target cells, moving from the cytoplasm to the nucleus. While delivering genes to the cytoplasm is well-established, the journey to the nucleus has been notoriously difficult. This inefficiency often necessitates high doses of DNA, which can provoke immune responses and cytotoxicity.
Innovative Approach to Gene Delivery
The new method leverages nuclear localization signals (NLS) — short peptide sequences that guide proteins into the nucleus. By attaching DNA to these NLS peptides, the DNA can ‘hitch a ride’ to the nucleus. Although this strategy has been explored for decades, results have been inconsistent and difficult to replicate.
One major obstacle has been the lack of advanced chemistry to observe and understand the DNA-NLS nuclear delivery process. Questions about the optimal length of NLS, the spacing between NLS and DNA, and the correct NLS sequence have remained unanswered. The Devaraj lab’s breakthrough provides a solution by developing a chemistry workflow that screens DNA-NLS conjugates, enabling researchers to identify the most effective configurations.
“In creating this workflow, we were able to conduct robust screens, essentially defining the design rules that allow you to attach one of these NLS peptides to a DNA gene cassette. We saw a greater than tenfold increase in nuclear DNA delivery,” stated Zulfiqar Mohamedshah, a biochemistry graduate student and first author of the paper.
How the Workflow Operates
The new workflow builds on an enzymatic DNA tagging technology, DNA-TAG, previously developed in the Devaraj Lab. The team utilized a bacteria-derived enzyme, TGT (tRNA guanine transglycosylase), to modify DNA with a chemical handle, facilitating the easy attachment of peptides, including NLS peptides, to DNA.
This process allowed the lab to modify DNA gene cassettes with NLS peptides, adjusting parameters such as the type of NLS used, the space between the NLS and the DNA, and the number of NLS attached. The gene cassette was encoded with an eGFP reporter that fluoresces green in human cells upon nuclear delivery and expression, allowing the team to screen various DNA-NLS conjugate permutations for effectiveness.
“We were able to get expression from the nuclear-targeted DNA greater than the expression of unmodified DNA at ten times the amount,” stated Devaraj, co-author and chair of the biochemistry department. “What this means is you can deliver less DNA to the cell while still increasing expression, which should mitigate cytotoxicity issues.”
Implications for Gene Therapy
The ultimate goal of gene therapy is to heal patients with genetic disorders. To test their workflow, the team delivered a gene cassette encoding Factor IX, a protein deficient in Christmas disease, a rare hereditary bleeding disorder. Their results demonstrated a tenfold higher expression of Factor IX compared to controls, underscoring the potential of DNA-NLS conjugates for non-viral gene therapy applications.
Moreover, the research is among the first to reveal that specific DNA-NLS sequences perform better in certain tissue types. For instance, hepatic tissue responded more favorably to certain NLS peptides than cardiac or renal tissue. This discovery opens the door for further research into tissue-specific DNA-NLS conjugate deployment.
The team plans to explore whether delivering DNA-NLS conjugates reduces immune responses, another challenge in gene therapy. They are also investigating the workflow’s application in enhancing genomic DNA edits using CRISPR-Cas-9, aiming to refine the process for clinical translation and scalability.
The study was authored by Zulfiqar Y. Mohamedshah, Chih-Chin Chi, Ember M. Tota, Alexis C. Komor, and Neal K. Devaraj, all affiliated with UC San Diego. Funding was provided in part by Seawolf Therapeutics, The Camille & Henry Dreyfus Foundation, and the National Institutes of Health.
