Home Systems biology New DNA repair kit successfully repairs inherited disease in patient-derived cells

New DNA repair kit successfully repairs inherited disease in patient-derived cells


Genetic mutations that cause debilitating inherited kidney disease affecting children and young adults have been fixed in patient-derived kidney cells using a potentially revolutionary DNA repair kit. The advance, developed by scientists at the University of Bristol, is published in Nucleic Acids Research.

In this new study, the international team describes how they created a DNA repair vehicle to genetically repair faulty podocin, a common genetic cause of hereditary steroid-resistant nephrotic syndrome (SRNS).

Podocin is a protein normally located on the surface of specialized kidney cells and essential for kidney function. The defective podocin, however, gets stuck inside the cell and never makes it to the surface, eventually damaging the podocytes. Since the disease cannot be cured with drugs, gene therapy that repairs the genetic mutations that cause faulty podocin offers hope for patients.

Typically, human viruses have been used in gene therapy applications to perform genetic repairs. These are used as a “Trojan horse” to enter the cells containing the errors. Currently dominant systems include lentiviruses (LV), adenoviruses (AV), and adeno-associated viruses (AAV), all of which are relatively harmless viruses that readily infect humans. However, these viruses all share the same limitation in that they are space-limited inside their viral shells. This in turn limits the amount of cargo they can deliver, namely the DNA kit necessary for efficient gene repair, which greatly limits the scope of their application in gene therapy.

Applying synthetic biology techniques, the team led by Dr Francesco Aulicino and Professor Imre Berger of Bristol’s School of Biochemistry, re-engineered baculovirus, an insect virus harmless to humans that is not no longer constrained by limited cargo capacity.

“What distinguishes baculovirus from LV, AV and AAV is the lack of a hard shell encapsulating the cargo space.” said Dr. Francesco Aulicino, who led the study. The baculovirus shell resembles a hollow stick – it simply lengthens as the cargo increases. This means that a much more sophisticated toolkit for repairing a genetic defect can be provided by the baculovirus, making it much more versatile than commonly used systems.

First, the baculovirus had to be equipped to enter human cells, which it normally would not. “We decorated the baculovirus with proteins that allowed it to enter human cells very efficiently.” explained Dr. Aulicino. This modified baculovirus is considered safe because it can only multiply in the insect, but not in human cells. The scientists then used their modified baculovirus to deliver much larger pieces of DNA than previously possible, and integrate them into the genomes of a range of human cells.

The DNA of the human genome comprises 3 billion base pairs making up ~25,000 genes, which code for proteins essential for cellular functions. If faulty base pairs occur in our genes, faulty proteins are made which can make us sick, resulting in an inherited disease. Gene therapy promises to repair inherited disease at its very root, by rectifying these errors in our genomes. Gene-editing approaches, particularly CRISPR/Cas-based methods, have significantly advanced the field by enabling gene repair with base-pair precision.

The team used podocytes derived from patients with the pathogenic error in the genome to demonstrate the suitability of their technology. By creating a DNA repair kit, comprising protein-based scissors and the nucleic acid molecules that guide them – and the DNA sequences to replace the faulty gene, the team delivered with a single modified baculovirus a healthy copy of the podocin gene along with the CRISPR/Cas machines to insert it with base-pair precision into the genome. This was able to reverse the pathogenic phenotype and restore podocin to the cell surface.

Professor Imre Berger explains: “We had previously used baculovirus to infect cultured insect cells to produce recombinant proteins to study their structure and function. This method, called MultiBac, developed by the Berger laboratory, has been very effective in making very large multiprotein complexes with many subunits, in laboratories around the world. “MultiBac was already exploiting the flexibility of the baculovirus shell to deliver large chunks of DNA into cultured insect cells, instructing them to assemble the proteins we were interested in.” When the scientists realized that the same property could potentially transform gene therapy in human cells, they set to work creating their new system described in their publication.

Dr. Aulicino added: “There are many ways to use our system. In addition to podocin repair, we were able to show that we can correct many errors at very different locations in the genome simultaneously, using our unique baculovirus delivery system and the newest editing techniques available.

“SRNS is one of the most common genetic diseases affecting the kidney,” said Professor Moin Saleem, a leading expert in gene therapy for inherited kidney disease at Bristol Renal. “SRNS is characterized by kidney failure at an early age, resulting in a severe loss of quality of life for those affected.

Professor Gavin Welsh, Professor of Renal Cell Biology at Bristol Renal, concluded: “These results are very encouraging. This novel approach developed by the Berger team holds promise not only for SRNS, but also for a range of other genetic kidney diseases, where efficient gene repair is not feasible with current technology. The implementation of a new vector system for clinical applications is still a long time, but we believe that the advantages offered make it a very interesting undertaking. »

This research has received funding from the European Research Council (ERC), Kidney Research UK (KRUK) and the EPSRC/BBSRC Bristol Research Center for Synthetic Biology BrisSynBio.


“Highly efficient CRISPR-mediated large-DNA docking and multiplexed primary editing using a single baculovirus” by F Aulicino et al. in nucleic acid research.