Gene therapy holds the promise of preventing and curing disease by manipulating gene expression within a patient's cells. However, to be effective, the new gene must make it into a cell's nucleus. The inability to consistently, efficiently do so has hampered progress in advancing treatment.
University of California San Diego researchers, led by Department of Biochemistry and Molecular Biophysics Professor Neal Devaraj's lab, have unveiled a new method that greatly increases the efficacy of gene delivery while minimizing harmful side effects to the cell. Their work appears in Nature Communications .
For gene therapeutics to be effective, the introduced gene must be delivered to target cells, ultimately moving from the cell's cytoplasm to the nucleus. While gene delivery into the cytoplasm is well-known and standardized, getting genes from the cytoplasm into the nucleus can be quite challenging.
To compensate for low nuclear translocation efficiency (estimated at around one percent), potential gene therapies can require very high doses of DNA to ensure an adequate amount reach the nucleus. These high doses can trigger immune responses and cytotoxicity.
Delivery of DNA into the nucleus can be done using nuclear localization signals (NLS) — short peptide sequences that act as molecular mail codes by tagging certain proteins for transport into the nucleus. Attaching the DNA to the NLS allows it to hitch a ride into the nucleus. Although this method has been in development for several decades, results thus far have been inconsistent and hard to reproduce.
This methodology has faced several challenges, the biggest of which was that, until now, the chemistry wasn't sufficiently developed to allow scientists to really observe and understand what happens during the DNA-NLS nuclear delivery. Does the length of the NLS matter? Does the space between the NLS and the DNA matter? Are researchers using the wrong NLS sequence? Should they attach multiple NLS to the DNA?
What was needed was a way to screen for all these variables so researchers could easily identify which permutations had the best results. This is exactly what the Devaraj lab created when they developed a chemistry workflow that can easily screen DNA-NLS conjugates, allowing users to define the parameters of the conjugations.
"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 It's Done
The new workflow was adapted from an enzymatic DNA tagging technology, DNA-TAG, previously developed in the Devaraj Lab. In this work, the team used a bacteria-derived enzyme TGT (tRNA guanine transglycosylase) to modify DNA with a chemical handle which allows for subsequent, easy attachment of peptides to DNA, including NLS peptides.
With this workflow, the lab was able to modify DNA gene cassettes — mobile snippets of DNA — with NLS peptides and then change the parameters of the NLS: the type of NLS used, the space between the NLS and the DNA, and the number of NLS attached to the DNA. The gene cassette was encoded with an eGFP reporter that fluoresces green in human cells upon nuclear delivery and expression.
In this way, they were able to screen different permutations of DNA-NLS conjugates to see which combinations were most effective at penetrating the nucleus. This new screening workflow allows researchers to precisely define and deploy the DNA-NLS conjugates with the highest nuclear delivery.
"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, one of the paper's co-authors 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."
The ultimate goal of any gene therapy is to heal sick patients, so 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 showed 10-fold higher expression of Factor IX than controls, highlighting the potential of DNA-NLS conjugates for non-viral gene therapy applications.
The paper is also one of the first to show that specific DNA-NLS sequences function better in specific tissue types: hepatic tissue had specific NLS peptides that were better for nuclear translocation than if used in cardiac or renal tissue. Further research could tease out how precisely these DNA-NLS conjugates can be deployed for tissue-specific delivery.
The team would like to further study whether delivering DNA-NLS conjugates to the cell decreases immune response — another hurdle with this kind of gene therapy. They're also investigating using the workflow on boosting genomic DNA edits using CRISPR-Cas-9, and hope to continue refining the workflow into something more clinically translatable and scalable, moving it closer to patient bedside.
Full list of authors: Zulfiqar Y. Mohamedshah, Chih-Chin Chi, Ember M. Tota, Alexis C. Komor and Neal K. Devaraj (all UC San Diego).
Funding provided, in part, from Seawolf Therapeutics, The Camille & Henry Dreyfus Foundation (ST-25-025) and the National Institutes of Health (R35GM141939 and T32GM146648).
