Periodic Reporting for period 2 - Cor-Edit-P (Cardiac open reading frame edition to study cardiomyopathies in pigs)
Período documentado: 2023-07-01 hasta 2024-12-31
Heart failure represents a leading cause of death in European societies. Besides ischemic and inflammatory causes, a growing number of patients are diagnosed with genetic mutations causing heart failure. Though pharmaceutical agents may be supportive in genetic heart disease, no causal treatment exists.
However, genetic editing has become available and is developed with rapid progress. About a decade after the landmark discovery of Nobel laureates Doudna and Charpentier that CRISPR-Cas9 can be adapted from the bacterial kingdom to work as applicable precision-guided genetic editors in mammalian cells, human diseases such as amyoidosis or sickle cell anemia or ß-thalassemia have been successfully treated in clinical studies. These developments open the door for a new class of therapeutics which are based on correction of disease-causing mutations in the genome, in cardiac as well as many other inherited diseases where no specific therapeutics are available. Exploiting the cutting-edge technology of gene editing, Cor-edit-P (Cardiac open reading frame edition to study cardiomyopathies in pigs) aims at specifically eliminating the underlying cause of genetic dilated cardiomyopathy (DCM) to improve cardiac function, reduce the risk of deadly arrhythmias and increase span and quality of life.
Cor-edit-P will
- generate currently lacking porcine models of genetic cardiomyopathy;
- exercise curative Crispr-Cas9 mediated gene editing of DCM in pigs in vivo, using the PLNR14del mutation in the phospholamban (PLN) gene as prominent example;
- use human patient-derived PLN-R14del ventricular progenitor cells for gene correction ex vivo followed by transplantation of corrected cells into PLN-R14del pigs.
Our approach implements a new paradigm for treating genetic cardiomyopathy and develops Crispr-Cas9 based gene therapy in pigs to foster clinical translation. Our work will influence the development of gene therapy by industry and academia and will benefit patients suffering genetic cardiomyopathy, but also further genetic diseases which are manifold prevalent in Europe.
However, genetic editing has become available and is developed with rapid progress. About a decade after the landmark discovery of Nobel laureates Doudna and Charpentier that CRISPR-Cas9 can be adapted from the bacterial kingdom to work as applicable precision-guided genetic editors in mammalian cells, human diseases such as amyoidosis or sickle cell anemia or ß-thalassemia have been successfully treated in clinical studies. These developments open the door for a new class of therapeutics which are based on correction of disease-causing mutations in the genome, in cardiac as well as many other inherited diseases where no specific therapeutics are available. Exploiting the cutting-edge technology of gene editing, Cor-edit-P (Cardiac open reading frame edition to study cardiomyopathies in pigs) aims at specifically eliminating the underlying cause of genetic dilated cardiomyopathy (DCM) to improve cardiac function, reduce the risk of deadly arrhythmias and increase span and quality of life.
Cor-edit-P will
- generate currently lacking porcine models of genetic cardiomyopathy;
- exercise curative Crispr-Cas9 mediated gene editing of DCM in pigs in vivo, using the PLNR14del mutation in the phospholamban (PLN) gene as prominent example;
- use human patient-derived PLN-R14del ventricular progenitor cells for gene correction ex vivo followed by transplantation of corrected cells into PLN-R14del pigs.
Our approach implements a new paradigm for treating genetic cardiomyopathy and develops Crispr-Cas9 based gene therapy in pigs to foster clinical translation. Our work will influence the development of gene therapy by industry and academia and will benefit patients suffering genetic cardiomyopathy, but also further genetic diseases which are manifold prevalent in Europe.
In particular, we have used the CRISPR-Cas9 nuclease system, which can break the DNA double strand at particular sites targeted by guide RNAs. We have developed Cas9 versions which are packable in small adeno-associated viruses (AAV9). Using this combination, we have achieved editing in cardiac tissue derived from pigs of up to 72% ex vivo. These vectors will be used for functional analysis in human organoids.
Furthermore, we have designed and generated a pig strain with a human mutation of a particular genetic cardiomyopathy, phospholamban-R14del. Heterozygous offspring of this cohort shows signs of arrhythmogenic potentials, corresponding to the affected patients. For this human mutation, we have developed a CRISPR-Cas9-derived editor, which can specifically affect the mutated, but not the healthy allele. This vector has already successfully tested in mice and will now be used in the new pig cohort with the human PLN R14del mutation.
Finally, we follow the strategy to embed new cardiac progenitor cells into the diseased myocardium of genetic cardiomyopathies. We have tested survival of a human ventricular progenitor (HVPs) cell source in pig hearts undergoing myocardial infarction and have established a functional benefit when applying these cells in this model. Next, we will apply these cells in a genetic cardiomyopathy model (PLN R14del).
Furthermore, we have designed and generated a pig strain with a human mutation of a particular genetic cardiomyopathy, phospholamban-R14del. Heterozygous offspring of this cohort shows signs of arrhythmogenic potentials, corresponding to the affected patients. For this human mutation, we have developed a CRISPR-Cas9-derived editor, which can specifically affect the mutated, but not the healthy allele. This vector has already successfully tested in mice and will now be used in the new pig cohort with the human PLN R14del mutation.
Finally, we follow the strategy to embed new cardiac progenitor cells into the diseased myocardium of genetic cardiomyopathies. We have tested survival of a human ventricular progenitor (HVPs) cell source in pig hearts undergoing myocardial infarction and have established a functional benefit when applying these cells in this model. Next, we will apply these cells in a genetic cardiomyopathy model (PLN R14del).
During the development of editors for PLN R14del, a novel strategy was rapidly established for full correction rather than elimination of the diseased allele: prime editing is combining Cas9 capability to interact with the DNA sequence targeted by a particular guide RNA and a reverse transcriptase. Cas9 activity in this case only concerns 1 of the 2 DNA strands (nicking) and is followed by reverse transcriptase inserting a sequence given by the pegRNA into the site nicked by Cas9. We have developed a prime editor correction the human PLN R14del mutation and have tested it ex vivo and in mice carrying the human mutation sequence (including surrounding regions). Here we were able to test our candidates and select the most successful with an ability to decrease the mutated PLN R14del transcript by 20% and the susceptibility to arrhythmias by >50%. We will refine this agent further and expect it to be applicable to the PLN R14del pig successfully, potentially offering specific cure to patients carrying this life-threatening mutation.