Recent advances in genetic engineering, by way of CRISPR mediated gene-editing technology, provide a method for creating genetic mutations associated withhuman disease conditions in animal models. The nonhuman primate more closely parallels humans allowing for the study of disease physiology to develop therapies and treatments of human diseases. We are using CRISPR/Cas9-mediated genome editing methods for three projects, as described below. Ultimately, the development of genome editing platforms in NHPs will enable us to create mutations that parallel human disease-associated mutations to better our understanding of disease progression and allow for development of treatments or therapeutics.
Introducing CCR5 Deletions Associated with HIV Resistance
Our first project focuses on genome editing in the cynomolgus monkey to generate null mutations in the CCR5 gene, which is associated with protection from, and cure of, HIV infection. These animals would then be able to serve as bone marrow transplant donors to experimentally test protection from AIDS in an NHP model. Using in vitro fertilization (IVF), we can introduce the CRISPR gene editing components into the early 1-cell monkey embryo, where the edits can be permanently created and maintained as the embryo continues to develop. We will assess embryo development outcomes through time-lapse embryo imaging. Edited embryos will then be transferred to a surrogate dam to initiate a pregnancy. We have successfully introduced deletions into the CCR5 gene of monkey embryos, and in the future, are planning to transfer embryos to surrogate dams to produce edited offspring.
Nikon TE-300 fluorescence microscope with micro-manipulators installed for embryo handling, IVF, ICSI, and gene injections.
Towards An Aromatase Deficiency Model
The CYP19A1 gene encodes for the aromatase enzyme which converts testosterone to estrogen in cells. CYP19A1 loss of function mutations in human patients are associated with aromatase deficiency, resulting in estrogen deficiency and hyperandrogenism. Our goal is to disrupt the CYP19A1 gene to create null mutations, similarly observed in human patients, to better understand the impact of estrogen deficiency on intrauterine fetal development, and on neurological, endocrine and metabolic function from birth to sexual maturation. We are currently testing the genome editing approach in rhesus macaque cells and look forward to genome editing in 1-cell embryos for future embryo transfer to surrogate dams.
Delivery of CRISPR gene-editing components to a marmoset zygote.
Correction of Disease Mutations for Developing Stem Cell Based Therapies of Frontotemporal Dementia
Instead of trying to create a disease-associated mutation, we aim to correct it! A small cohort of rhesus macaques in the Wisconsin National Primate Research Center contain a mutation in the MAPT gene, which is associated with frontotemporal dementia in human patients. We have isolated cells from the monkeys containing the mutation to serve as an in vitro platform for evaluating the role of the mutation and testing the approach to correct the mutation. First, we can reprogram these cells to induced pluripotent stem cells (iPSCs) and then differentiate them to neurons in a dish to assess physiological differences between MAPT mutant cells compared to control, wild-type cells. Second, we can then test the editing approach to correct the mutation and determine the impact of the correction on cell function. The cellular editing platform will serve as a foundation for developing cell and gene therapies in NHPs, with ultimate translation to human patients.