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New technology that can rewrite genetic code raises hopes for gene therapy — ScienceDaily

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Genetic mutations that cause a debilitating inherited kidney disease that affects children and young adults have been fixed in patients’ kidney cells using a potentially game-changing DNA repair kit. The advance, developed by scientists at the University of Bristol, was published in the Nucleic acid research.

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

Podacin is a protein that is normally found on the surface of specialized kidney cells and is essential for kidney function. The faulty podocin, however, remains inside the cell and never reaches the surface, ultimately damaging the podocytes. Because the disease cannot be cured with drugs, gene therapy, which reverses the genetic mutations that cause the faulty podocin, offers hope for patients.

As a rule, human viruses are used in gene therapy to carry out genetic repairs. They are used as a Trojan horse to enter cells with errors. Currently, the dominant systems include lentivirus (LV), adenovirus (AV), and adeno-associated virus (AAV), which are relatively harmless viruses that readily infect humans. However, all of these viruses share the same limitation in that they are confined to the space within their viral envelopes. This, in turn, limits the amount of cargo they can deliver, namely the set of DNA required for efficient genetic repair, which greatly limits their scope in gene therapy.

Using synthetic biology techniques, a team led by Dr Francesco Aulicino and Professor Imre Berger from the Bristol School of Biochemistry has re-engineered baculavirus, a harmless insect virus that is no longer limited by its limited payload.

“What distinguishes baculavirus from LV, AV, and AAV is the lack of a hard envelope that seals the cargo space.” – said Dr. Francesco Aulichino, who led the study. The baculovirus envelope resembles a hollow stick – it just gets longer as the load increases. This means that the baculavirus can create a much more sophisticated toolkit for correcting the genetic defect, making it far more versatile than commonly used systems.

First, the baculavirus had to be equipped to enter human cells, which it normally did not do. “We decorated the baculavirus with proteins that allowed it to enter human cells very efficiently.” – explained Dr. Avlitsyna. This modified baculovirus is considered safe because it can only reproduce in insects, but not in human cells. The scientists then used an engineered baculavirus to deliver much larger pieces of DNA than previously possible and insert them into the genome of a range of human cells.

The DNA in the human genome consists of 3 billion base pairs, which make up ~25,000 genes that encode proteins required for cellular functions. When faulty base pairs occur in our genes, faulty proteins are made that can make us sick, leading to inherited diseases. Gene therapy promises to reverse inherited disease at its very root by correcting such errors in our genomes. Gene editing approaches, particularly CRISPR/Cas-based methods, have greatly advanced the field, enabling genetic repair down to base pairs.

To demonstrate the applicability of their technology, the team used podocytes obtained from patients who carry a disease-causing error in their genome. By creating a DNA repair kit containing scissor proteins and the nucleic acid molecules that guide them, as well as DNA sequences to replace the faulty gene, the team used a single engineered baculavirus to deliver a healthy copy of the podocin gene, which combines with CRISPR/Cas equipment to insert it into the genome to the nearest base pair. This was able to reverse the disease-causing phenotype and restore podacin to the cell surface.

Professor Imre Berger explains: “Previously, we used baculavirus to infect cultured insect cells to produce recombinant proteins to study their structure and function.” This method, called MultiBac, developed by Berger’s lab, has been very successful for generating very large multiprotein complexes with many subunits in laboratories around the world. “MultiBac has already used the flexibility of the baculavirus envelope to deliver large pieces of DNA into cultured insect cells, telling 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 about creating the new system described in their publication.

Dr. Aulichyna added, “There are many ways to use our system. In addition to restoring podocin, we can show that we can simultaneously efficiently correct many errors at very different locations in the genome using our single baculavirus delivery system and the latest available editing techniques.”

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

Professor Gavin Welsh, Professor of Renal Cell Biology at the Bristol Renal Unit, concluded: ‘These results are very encouraging. This new approach, founded by Berger’s team, is promising not only for SRNS, but also for a number of other genetic kidney diseases, where effective genetic repair is not possible with current technologies. There is a long way to go before a new vector system can be implemented for clinical use, but we believe that the advantages offered make this a very worthwhile endeavor.”

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

*About steroid-resistant nephrotic syndrome (SRNS)

Hereditary CRNS is a devastating kidney disease that affects children and young adults. It is caused by mutations in genes that are important for the functioning of a specialized filtering cell in the kidney called a podocyte. These disorders are currently untreatable and lead to rapid onset of renal failure, requiring dialysis and transplantation.

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