Developing CRISPR-Cas gene therapy for monogenic immune diseases
This study aims to construct a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) based gene therapy platform for monogenic immune diseases. We will test three different CRISPR platforms for stem cell editing: standard, prime and custom CRISPR. The stem cells are first harvested from healthy human donors, edited with one of the aforementioned platforms, and thereafter infused to recipient mice. The goal is to i) evaluate engraftment and persistence of the edited cells, ii) compare the safety and efficacy of the different editing systems. In addition, we will evaluate new approaches to preserve stem cell pluripotency during editing, when they are published.
We want to conduct this research on two mouse models that are suitable for human stem cell engraftments:
1. NOD.Cg-KitW-41J Tyr+ Prkdcscid Il2rgtm1Wjl (NBSGW): this mice do not need pre-conditioning prior cell transplant however, this model is new and perhaps may not reflect the whole situation during the bone marrow transplantation in humans. Therefore we want to also include second mouse model:
2. NOD.Cg-Prkdcscid IL2rgtmlWjl/Sz (NSG): these mice need conditioning prior human cell transplant. We chose the most popular and well established conditioning: total body irradiation (TBI) and will run pilot test to establish protocols. The irradiation treatment is non-invasive. Animals are not observed to display any signs of acute pain associated with the procedure though general
malaise in the post-irradiation period is common. Not all rodents respond equally to radiation and some death is expected to occur, therefore the estimated number of animals accounts for animal death. Our goal is not to proceed with this treatment if in pilot experiments we will experience excessive animal death.We expect mild and mild to moderate distress in Model 1 and Model 2, respectively. We will also breed these two animal models in the facility, therefore the estimated number of animals is 3972. We will breed animals as homozygotes, which meas all the offspring will be used in experiments or to replace breeders. We will always run pilot experiments to test the protocols but most importantly to evaluate the animal welfare. We will prioritize the experiments in Model 1 which requires lower amount of animals.
This research project plans to test editing in 4 different human loci using 3 different CRISPR strategies in order to find the best one that will be the most precise and will cause the lest to none off-target effects which is crucial when translating this technology to clinical use.
We want to conduct this research on two mouse models that are suitable for human stem cell engraftments:
1. NOD.Cg-KitW-41J Tyr+ Prkdcscid Il2rgtm1Wjl (NBSGW): this mice do not need pre-conditioning prior cell transplant however, this model is new and perhaps may not reflect the whole situation during the bone marrow transplantation in humans. Therefore we want to also include second mouse model:
2. NOD.Cg-Prkdcscid IL2rgtmlWjl/Sz (NSG): these mice need conditioning prior human cell transplant. We chose the most popular and well established conditioning: total body irradiation (TBI) and will run pilot test to establish protocols. The irradiation treatment is non-invasive. Animals are not observed to display any signs of acute pain associated with the procedure though general
malaise in the post-irradiation period is common. Not all rodents respond equally to radiation and some death is expected to occur, therefore the estimated number of animals accounts for animal death. Our goal is not to proceed with this treatment if in pilot experiments we will experience excessive animal death.We expect mild and mild to moderate distress in Model 1 and Model 2, respectively. We will also breed these two animal models in the facility, therefore the estimated number of animals is 3972. We will breed animals as homozygotes, which meas all the offspring will be used in experiments or to replace breeders. We will always run pilot experiments to test the protocols but most importantly to evaluate the animal welfare. We will prioritize the experiments in Model 1 which requires lower amount of animals.
This research project plans to test editing in 4 different human loci using 3 different CRISPR strategies in order to find the best one that will be the most precise and will cause the lest to none off-target effects which is crucial when translating this technology to clinical use.