A potentially game-changing DNA repair kit was used to repair genetic mutations that cause a debilitating hereditary kidney disease affecting children and young adults in patient-derived kidney cells.

DNA repair kit
Image of patient-derived podocyte kidney cells repaired using the Berger team’s novel baculovirus-vectored approach. Podocin (green) is restored to the cell surface, as it is in healthy podocytes.
Francesco Aulicino of the University of Bristol

A potentially game-changing DNA repair kit was used to repair genetic mutations that cause a debilitating hereditary kidney disease affecting children and young adults in patient-derived kidney cells. The breakthrough, developed by scientists at the University of Bristol, has been published in Nucleic Acids Research.

The international team describes how they developed a DNA repair vehicle to genetically repair faulty podocin, a common genetic cause of inheritable Steroid Resistant Nephrotic Syndrome (SRNS).

Podocin is a protein found on the surface of specialized kidney cells that is required for kidney function. Faulty podocin, on the other hand, remains trapped inside the cell and never reaches the surface, causing podocytes to die. Because medications cannot cure the disease, gene therapy, which repairs the genetic mutations that cause the faulty podocin, offers patients hope.

Human viruses have traditionally been used in gene therapy applications to perform genetic repairs. These are used as a “Trojan Horse” to enter cells containing errors. Lentivirus (LV), adenovirus (AV), and adeno-associated virus (AAV) are currently dominant systems. These viruses are all relatively harmless and easily infect humans. These viruses, however, all have the same limitation in that they are limited in space within their viral shells. This, in turn, limits the amount of cargo they can deliver, namely the DNA kit required for efficient genetic repair, severely limiting their application in gene therapy.

The team led by Dr. Francesco Aulicino and Professor Imre Berger from Bristol’s School of Biochemistry used synthetic biology techniques to re-engineer baculovirus, a harmless insect virus for humans that is no longer constrained by cargo capacity.

“What distinguishes baculovirus from LV, AV, and AAV is the absence of a rigid shell enclosing the cargo space.” Dr. Francesco Aulicino, who led the study, said Baculovirus’s shell is similar to a hollow stick; it simply grows longer as the cargo increases. This means that the baculovirus can deliver a much more sophisticated toolkit to repair a genetic defect, making it far more versatile than commonly used systems.

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

The human genome’s DNA is made up of 3 billion base pairs that make up 25,000 genes that encode proteins that are required for cellular functions. When faulty base pairs occur in our genes, faulty proteins are produced, which can cause us to become ill and result in hereditary disease. By correcting such errors in our genomes, gene therapy promises to repair hereditary illness at its root. Gene editing approaches, particularly CRISPR/Cas-based methods, have greatly advanced the field by enabling base-pair precision genetic repair.

To demonstrate the capability of their technology, the researchers used patient-derived podocytes with the disease-causing error in the genome. The team delivered a healthy copy of the podocin gene along with the CRISPR/Cas machinery to insert it with base-pair precision into the genome using a single engineered baculovirus and a DNA repair kit comprised of protein-based scissors and the nucleic acid molecules that guide them — and the DNA sequences to replace the faulty gene. This was successful in reversing the disease-causing phenotype and reintroducing podocin to the cell surface.

“We had previously used baculovirus to infect cultured insect cells to produce recombinant proteins for studying their structure and function,” explains Professor Imre Berger. This method, known as MultiBac, was developed by the Berger laboratory and has been used successfully in laboratories all over the world to create very large multiprotein complexes with many subunits. “MultiBac had already used the baculovirus shell’s flexibility to deliver large pieces of DNA into cultured insect cells, instructing them to assemble the proteins we were interested in.” When the researchers realized that the same property could potentially transform gene therapy in human cells, they set to work developing their new system, which they describe in their paper.

Dr. Aulicino continued: “There are numerous ways to put our system to use. In addition to podocin repair, we were able to demonstrate that by using our single baculovirus delivery system and the most recent editing techniques available, we could efficiently correct many errors in very different places in the genome.”

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

Professor Gavin Welsh, Professor of Renal Cell Biology at Bristol Renal, came to the following conclusion: “These findings are extremely encouraging. The Berger team’s new approach holds promise not only for SRNS but also for a variety of other genetic kidney diseases where efficient genetic repair is not possible with current technology. It will be a long road to implement a new vector system for clinical applications, but we believe the benefits will be well worth the effort.”

The European Research Council (ERC), Kidney Research UK (KRUK), and the EPSRC/BBSRC Bristol Research Centre for Synthetic Biology BrisSynBio funded this study.

*Information on Steroid Resistant Nephrotic Syndrome (SRNS)

Hereditary SRNS is a kidney disease that primarily affects children and young adults. It is caused by mutations in genes involved in the function of the podocyte, a specialized filtration cell of the kidney. There is currently no treatment for these disorders, which cause rapid onset of kidney failure and necessitate dialysis and transplantation.

Source: Materials provided by the University of Bristol.

Reference: DOI: 10.1093/nar/gkac587

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