Periodic Reporting for period 1 - PRIME (Prime editing to Repair Inherited Metabolic Errors: in vivo gene correction for human genetic disease)
Periodo di rendicontazione: 2022-09-01 al 2025-02-28
Metabolic diseases are generally severe genetic diseases without good treatment options. While individual metabolic diseases are rare, as a group, they affect many patients an represent one of the major causes of childhood death in Europe. Methylmalonic acidemia (MMA) is an example of such a rare genetic metabolic disorder. MMA affects the body’s ability to break down specific amino acids. Even when strictly adhering to current therapeutic strategies, patients may still develop metabolic decompensations, neurologic symptoms, kidney failure, and acute blindness. Liver transplantation can prevent further accumulation of toxic metabolites, but is a high-risk operation for MMA patients and requires lifelong immune suppression. Correction of the genetic cause of disease would revolutionize outcomes for these patients, especially how that MMA is increasingly detected in neonatal screening programs, which allows identification of patients prior to the onset of irreversible damage. However, efficiency, safety, versatility and delivery of precise gene editing is currently insufficient for clinical gene-correction therapies.
The aim of this proposal is to develop an in vivo gene-editing therapy program targeting the liver for patients with metabolic disease. As a proof-of-principle, we will repair genetic causes of MMA, validate this in personalized cell and mouse models, and lay the groundwork for human trials.
To this end, I will:
(1) Generate a clinically applicable gene-editing system. We will employ prime editing, a novel gene-editing technique, we recently demonstrated to accurately correct different mutations in patient-derived cell models. Using an innovative reporter and patient-derived cell models, I will develop prime editing into an efficient and safe strategy to correct >90% of patient mutations.
(2) Create a delivery system for the large prime-editing machinery with mRNA-packed lipid nanoparticles to target the liver.
(3) Develop a roadmap for tailored gene-correction therapies for a common human MMA mutation.
This foundational work will lay the basis for broad clinical application of precise gene-editing therapies.
The aim of this proposal is to develop an in vivo gene-editing therapy program targeting the liver for patients with metabolic disease. As a proof-of-principle, we will repair genetic causes of MMA, validate this in personalized cell and mouse models, and lay the groundwork for human trials.
To this end, I will:
(1) Generate a clinically applicable gene-editing system. We will employ prime editing, a novel gene-editing technique, we recently demonstrated to accurately correct different mutations in patient-derived cell models. Using an innovative reporter and patient-derived cell models, I will develop prime editing into an efficient and safe strategy to correct >90% of patient mutations.
(2) Create a delivery system for the large prime-editing machinery with mRNA-packed lipid nanoparticles to target the liver.
(3) Develop a roadmap for tailored gene-correction therapies for a common human MMA mutation.
This foundational work will lay the basis for broad clinical application of precise gene-editing therapies.
(1) Generate a clinically applicable gene-editing system:
During this project many different new prime editors have evolved. We have tested these for our target mutations, which imrpoved versatility and efficiency of gene correction. To further improve prime-editing efficiency for the many different mutations, we are personalizing pegRNA design in combination with the different prime editors. We are currently preparing for a large AI-leveraged CRISPR-screen to test >1000 prime-editor and pegRNA combinations for our mutations of interest. The results of this screen will not only yield optimized prime-editing tools for our mutations of interest, but will also be used to develop a prime editor/pegRNA design approach to tailor the prime-editing tools to any mutation of interest.
(2) Create a delivery system for the large prime-editing machinery with mRNA-packed lipid nanoparticles to target the liver.
We have optimized the systems to generate modRNA from the prime editors and pegRNAs and have tested and optimized different lipids to encapsulate these prime-editing tools in lipid nanoparticles. Using the optimized conditions, we have performed the first pilot experiments in a MMA-mouse model. Although the procedure was successful, this did not result in efficient gene editing in the mice. We are currently optimizing our tools in the CRISPR-screen (see obj. 1) and optimizing mouse-derived in vitro models to prepare for further experiments in the mice.
(3) Develop a roadmap for tailored gene-correction therapies for a common human MMA mutation.
Although this part of the project still has to be performed (from 30 months onwards), we have invested in further developing the roadmap for clinical gene-correction therapies by applying for an ERC-POC grant, which was granted. This will allow us to investigate the regulatory, financial, manufacturing and ethical requirements to develop these revolutionary therapies in a feasible, morally sound manner. Moreover, I have gained clinical experience with treating the first MMA patients in the Netherlands with mRNA of the deficient MUT-enzyme as the principal investigator of an industry-led trial.
Main achievements:
- Optimization of the prime-editing tools and preparations of an AI-leveraged large CRISPR-screen
- Generation of a delivery system for the prime-editing tools targeting the liver
- New grants (ERC-POC 2024; KNAW Ammodo Science Award for Groundbreaking Science; ZonMW VICI grant (to develop gene-editing therapies beyond the liver) and a TKI Health Holland grant for an in vivo model system to test delivery of gene-editing tools to different organs, the UMCU Team Award (TEAmRNA) and an NWA-ORC Nanospresso grant (as co-applicant) to develop bedside production of nanomedicines.
- a full professor position
- treatment of the first Dutch patients with systemically delivered mRNA in lipid nanoparticles in the Moderna Phase I/II MMA-mRNA clinical trial to treat MMA-patients with mRNA of the deficient Mut-enzyme.
During this project many different new prime editors have evolved. We have tested these for our target mutations, which imrpoved versatility and efficiency of gene correction. To further improve prime-editing efficiency for the many different mutations, we are personalizing pegRNA design in combination with the different prime editors. We are currently preparing for a large AI-leveraged CRISPR-screen to test >1000 prime-editor and pegRNA combinations for our mutations of interest. The results of this screen will not only yield optimized prime-editing tools for our mutations of interest, but will also be used to develop a prime editor/pegRNA design approach to tailor the prime-editing tools to any mutation of interest.
(2) Create a delivery system for the large prime-editing machinery with mRNA-packed lipid nanoparticles to target the liver.
We have optimized the systems to generate modRNA from the prime editors and pegRNAs and have tested and optimized different lipids to encapsulate these prime-editing tools in lipid nanoparticles. Using the optimized conditions, we have performed the first pilot experiments in a MMA-mouse model. Although the procedure was successful, this did not result in efficient gene editing in the mice. We are currently optimizing our tools in the CRISPR-screen (see obj. 1) and optimizing mouse-derived in vitro models to prepare for further experiments in the mice.
(3) Develop a roadmap for tailored gene-correction therapies for a common human MMA mutation.
Although this part of the project still has to be performed (from 30 months onwards), we have invested in further developing the roadmap for clinical gene-correction therapies by applying for an ERC-POC grant, which was granted. This will allow us to investigate the regulatory, financial, manufacturing and ethical requirements to develop these revolutionary therapies in a feasible, morally sound manner. Moreover, I have gained clinical experience with treating the first MMA patients in the Netherlands with mRNA of the deficient MUT-enzyme as the principal investigator of an industry-led trial.
Main achievements:
- Optimization of the prime-editing tools and preparations of an AI-leveraged large CRISPR-screen
- Generation of a delivery system for the prime-editing tools targeting the liver
- New grants (ERC-POC 2024; KNAW Ammodo Science Award for Groundbreaking Science; ZonMW VICI grant (to develop gene-editing therapies beyond the liver) and a TKI Health Holland grant for an in vivo model system to test delivery of gene-editing tools to different organs, the UMCU Team Award (TEAmRNA) and an NWA-ORC Nanospresso grant (as co-applicant) to develop bedside production of nanomedicines.
- a full professor position
- treatment of the first Dutch patients with systemically delivered mRNA in lipid nanoparticles in the Moderna Phase I/II MMA-mRNA clinical trial to treat MMA-patients with mRNA of the deficient Mut-enzyme.
- the prime-editor and pegRNA optimizations resulting from the insights from our CRISPR-screen may yield innovations that are beyond the state of the art. We are currently exploring the first promising strategies in different cell models and found improved prime-editing efficiency (for most .
- to improve functional analysis of prime-editing effects, we improved hepatic differentiation of adult stem cell derived liver organoids (patent: 24173809.5) because we had found that our liver organoids resembled biliary rather then liver cells (Ardisasmitta, Commun Biol 2022). Based on these insights, we tried to improve the liver phenotype with a different culturing system. This yielded unexpectedly liver-like metabolic activity for several pathways, including energy metabolism and drug metabolism. This new liver model which we hence called Hepatocyte-like Liver Organoids (HeLLO) may change the field of toxicity testing during drug development. We already won several prizes with the HeLLOs (the Ureka Challenge, a RegMed Voucher; the Utrecht Holdings Voucher, the Animal Free Venture Challenge, the BiotechBooster) and made industrial connections to start valorization.
- treatment of human MMA patients with mRNA in lipid nanoparticles: Although the effects of the mRNA therapy are short-lived and patients require infusions every 2 weeks, this first-in-human study with mRNA/LNPs for MMA does not only present a new treatment modality for MMA and other liver-dominant metabolic diseases, but this also provides a crucial proof-of-concept for the feasibility to temporarily express a protein (like the prime-editor) in the human liver using this delivery strategy. This serves as an important stepping stone for the development of in vivo prime-editing therapies.
- to improve functional analysis of prime-editing effects, we improved hepatic differentiation of adult stem cell derived liver organoids (patent: 24173809.5) because we had found that our liver organoids resembled biliary rather then liver cells (Ardisasmitta, Commun Biol 2022). Based on these insights, we tried to improve the liver phenotype with a different culturing system. This yielded unexpectedly liver-like metabolic activity for several pathways, including energy metabolism and drug metabolism. This new liver model which we hence called Hepatocyte-like Liver Organoids (HeLLO) may change the field of toxicity testing during drug development. We already won several prizes with the HeLLOs (the Ureka Challenge, a RegMed Voucher; the Utrecht Holdings Voucher, the Animal Free Venture Challenge, the BiotechBooster) and made industrial connections to start valorization.
- treatment of human MMA patients with mRNA in lipid nanoparticles: Although the effects of the mRNA therapy are short-lived and patients require infusions every 2 weeks, this first-in-human study with mRNA/LNPs for MMA does not only present a new treatment modality for MMA and other liver-dominant metabolic diseases, but this also provides a crucial proof-of-concept for the feasibility to temporarily express a protein (like the prime-editor) in the human liver using this delivery strategy. This serves as an important stepping stone for the development of in vivo prime-editing therapies.