Correlating molecular behavioral phenotypes in a marmoset model of Huntingtons disease

ABSTRACT
The common marmoset provides a very relevant primate model for understanding the organization of the human
nervous system and the diseases that affect it. Like humans, marmosets also demonstrate cooperative social
behavior and have advanced cognitive processes, making them of great interest in the field for modeling
developmental and psychiatric diseases and their therapies. They are also ideal for multigenerational genetic
experiments as they give birth twice a year and mature faster than most primates. However, while the
CRISPR/Cas9 system has been used to knockout genes and create knock-ins of single amino acids in a heritable
manner in marmosets, it has been a challenge in the field to create germline transmissible models of gene
reporters and trinucleotide repeat genes analogous to their murine counterparts. The very low efficiency of
homologous recombination (HR) in primates has precluded knocking-in coding sequences by simply injecting
Cas9 protein and a guide RNA into embryos during in vitro fertilization (IVF) as is done for creating knockouts.
This limitation has prevented modelling of more genetically complex neurological diseases such as Huntington’s
disease (HD) and for creating conditional reporters in marmosets, both of which are mainstays in the mouse
neurogenetics field. In addition to low HR frequency, other barriers to creating germline transmission of knock-
ins include the absence of a well annotated marmoset genome until recently, lack of protocols for derivation of
ground state marmoset pluripotent stem cells (cjPSCs), the low percentage of marmoset pregnancies after
embryo reimplantation, and a general deficiency of developmental biology expertise in the marmoset field. We
propose to harness our labs’ expertise in developmental biology, IVF technologies, and transgenic stem cell
biology to overcome this barrier to widespread use of marmosets. We aim to create transgenic knock-in cjPSCs,
convert them into ground-state pluripotent stem cells and then inject them into IVF morula to create a chimeric
founder marmoset that carries the modified genome. We then aim to screen the transgenic gametes from the
founder marmosets to create the F1 progeny and use them to correlate the molecular-behavioral phenotype of
HD. As proof-of-principle, we will focus on three knock-in reporter lines to broadly target excitatory, inhibitory,
and peripheral neuronal populations. Together, if successful, our aims will result in creation of the first primate
model with neuron-specific reporters, establish the marmoset as a valid model of HD, enable access to single-
cell transcriptomic changes at the early stages of HD in a primate disease model, and finally correlate these
molecular changes with the behavioral phenotype. These aims will provide fundamental insights into the biology
of HD and the role of huntingtin protein in different classes of neurons. The outcome of this project will also
influence a better understanding of poly-glutamine neurodegenerative diseases that affect humans. In addition,
the transgenic marmosets that we generate will be broadly available to the research community and enable new
studies into neural circuits, development, behavior, and a wide range of optogenetic applications.

Grant Number: 5U01DA054182-05
NIH Institute/Center: NIH
Principal Investigator: ALI BRIVANLOU