Gene mutations that cause debilitating hereditary kidney disease in children and young adults are fixed in patient-derived kidney cells. Using a potentially game-changing DNA repair kit. The progress, developed by University of Bristol scientists, has been published in nucleic acid research.
In this new study, the international team describes how they devised a DNA repair method to repair genetically faulty podocin, a common genetic cause of steroid-resistant nephrotic syndrome (SRNS).
Podocin is a protein normally found on the surface of specialized kidney cells and is essential for kidney function. However, the defective podocin remains stuck inside the cell and never reaches the surface, permanently damaging the podocin cells. Because the disease cannot be cured with medication, gene therapy that repairs the gene mutations that cause faulty podocin offers hope to patients.
Human viruses are commonly used in gene therapy applications to make genetic repairs. These are used as a “Trojan horse” to enter cells that carry errors. Currently dominant systems include lentivirus (LV), adenovirus (AV), and adeno-associated virus (AAV), which are all relatively harmless viruses that easily infect humans. However, these viruses all share the same limitation in that they are confined to the space within their viral envelope. This in turn limits the amount of charge they can deliver, that is, the pool of DNA required for effective genetic repair, greatly limiting the scope of their application in gene therapy.
By applying synthetic biology techniques, the team was led by Dr. Francesco Olicino and Professor Emre Berger From the Bristol School of Biochemistry, re-engineered baculovirus, an insect virus harmless to humans no longer constrained by limited cargo capacity.
“What distinguishes a bacterial virus from LV, AV, and AAV is the lack of a hard shell that encapsulates the cargo space.” said Dr. Francesco Olicino, who led the study. The shell of a bacterial virus is like a hollow stick – it simply becomes longer when the load increases. This means that a more complex set of genetic defect repair tools can be delivered by the bacterial virus, making them more diverse than commonly used systems.
First, the bacterial virus had to be prepared to penetrate human cells which it usually does not do. “We decorated the bacterial virus with proteins that enabled it to enter human cells very efficiently.” Dr. Ulysino explained. This modified bacterial virus is considered safe, as it can reproduce only in the insect, but not in human cells. The scientists then used the engineered bacterial virus to attach much larger pieces of DNA than was previously possible, and build these pieces into the genomes of an entire group of human cells.
The DNA in the human genome is made up of 3 billion base pairs that make up about 25,000 genes, which encode proteins essential for cellular functions. If defective base pairs occur in our genes, defective proteins are made that can make us sick, resulting in a genetic disease. Gene therapy promises to repair genetic diseases at their root, by correcting such errors in our genomes. Gene editing approaches, in particular CRISPR/Cas-based methods, have advanced the field significantly by enabling gene repair with base pair precision.
The team used patient-derived podocytes carrying the disease-causing error in the genome to demonstrate the efficacy of their technique. By creating a DNA repair kit, which includes protein-based scissors, the DNA molecules that guide them – and DNA sequencing to replace the faulty gene, the team provided with a single engineered baculovirus a healthy copy of the Podocin-associated gene for CRISPR/Cas machinery to precisely insert the base pair. in the genome. This was able to reverse the pathogenic phenotype and restore podocin to the cell surface.
Mr “We have previously used the bacterial virus to infect cultured insect cells to produce recombinant proteins to study their structure and function,” explains Emery Berger. This method, called MultiBac, developed by Berger’s lab, has been very successful in creating very large multiprotein complexes with many subunits, in laboratories around the world. “MultiBac really took advantage of the flexibility of the bacterial virus envelope to deliver large pieces of DNA into cultured insect cells, instructing them to synthesize the proteins we were interested in.” When the scientists realized that the same property could alter gene therapy in human cells, they went to work to create their new system described in their publications.
Dr. Ulysino added: “There are many avenues to benefit from our system. In addition to podocin repair, we can demonstrate that we can simultaneously correct many errors at very different places in the genome efficiently, using a single bacterial virus delivery system and the latest editing techniques available.”
“SRNS is one of the most common genetic diseases that affect the kidneys,” he said. Professor Moeen SaleemLeading expert in gene therapy for hereditary kidney disease at Bristol Raynal. “Acute respiratory sexual therapy is characterized by renal failure at an early age, which results in a massive loss of quality of life for sufferers.”
Professor Gavin WelchProfessor of Renal Cell Biology at Bristol Renal concluded: “These results are very encouraging. This new approach pioneered by Berger’s team holds promise not only for SRNS, but also for a range of other genetic diseases of the kidney, where effective genetic repair is not possible with current technology.” It is a long way forward to implement a new vector system for clinical applications, but we believe that the advantages presented make this a worthwhile task.”
This research received funding from the European Research Council (ERC), Kidney Research UK (KRUK), and the Bristol EPSRC/BBSRC Research Center for Synthetic Biology BrisSynBio.
“High-efficiency CRISPR-mediated large-scale DNA docking and multiplexed primary editing using a single baculovirusWritten by F Aulicino et al. in Nucleic acid research.
Notes to editors:
For more information or to arrange an interview with Professor Emre BergerAnd the Please contact firstname.lastname@example.org [Mon/Tues]Or send an email to email@example.com or Caroline Clancy [Wed to Fri]email firstname.lastname@example.org Mobile: +44 (0) 7776 170238 at the University of Bristol.
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Image is available for download over here.
Photo Caption: An image of kidney cone cells taken from a patient that were repaired using a new baculovirus-directed approach devised by Berger’s team. Podocin (stained in green) is restored to the cell surface as in healthy podocytes.
Credit: Dr. Francesco Olesino, University of Bristol.
About Professor Emery Berger FMedSci
Emery Berger is also a Director The Max Planck Bristol Center for Minimal Biology, director of the Center for Synthetic Biology at Bristol BrisSynBio, co-director of Bristol Biodesign Instituteand Wellcome Trust Investigator and ERC Investigator.
About Professor Moeen Selim
Moin Saleem is the director of Bristol Raynal, a pediatric nephrologist at Bristol Royal Children’s Hospital. He is also the co-founder and CSO of Purespring Therapeutics, a kidney gene therapy company.
About Professor Gavin Welch
Gavin Welch is Professor of Renal Cell Biology at Bristol Renal and Co-Founder and Scientific Director of Purespring Therapeutics.
About the Max Planck Center Bristol
The Max Planck Center Bristol (MPBC) is a joint research center of Max Planck Society The University of Bristol. MPBC focuses on the field of synthetic and minimal biology. Located in Bristol with contract at the Max Planck Institutes in Martinsried, Mainz and Heidelberg, scientists at MPBC aim to build artificial cells, cytoskeletons and nanoscale molecular machines to explore the building blocks of life and their applications.
About Steroid Resistant Nephrotic Syndrome (SRNS) SRNS is a devastating kidney disease that affects children and young adults. It is caused by mutations in genes important in the function of a specialized filtering cell in the kidney, called the podocyte. There is currently no cure for these disorders, and they lead to rapid onset of kidney failure, requiring dialysis and transplantation.
About Purespring Therapeutics
Purespring treatments It is an affiliate of the University of Bristol, and aims to deliver gene therapy to podocytes in order to treat previously incurable forms of kidney disease. It is funded by a £45 million investment from biotech company Syncona Venture Capital.
Published by the University of Bristol.
nucleic acid research
High-efficiency CRISPR-mediated large-scale DNA docking and multiplexed primary editing using a single baculovirus
The date the article was published
July 22 2022