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Generation Of Animal Models For Arteriovenous And Capillary Malformation
Patrick J. Smits, Dennis J. Konczyk, Christopher L. Sudduth, Jeremy A. Goss, Arin K. Greene.
Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

PURPOSE: Arteriovenous malformation (AVM) and capillary malformation (CM) are sporadic, non-hereditary, vascular malformations. AVMs and CMs enlarge over time and cause disfigurement, ulceration, pain, bleeding, infection and even heart failure and death. Drugs for AVM or CM do not exist. Activating mutations in MAP2K1 and GNAQ in endothelial cells (ECs) are causative for AVM and CM respectively. Our goal is to generate mouse models that will help develop therapies for AVM and CM.
METHODS: The most common AVM mutation (p.K57N) is located in exon 2 of the MAP2K1 gene, while the CM mutation (p.R183Q) is located in exon 4 of the GNAQ gene. Using CRISPR/Cas9 gene editing, we introduced these mutations into the mouse genome by replacing the wild-type exon (2 or 4) of one MAP2K1 or GNAQ allele with a mutant exon (Fig. 1 shows the strategy for MAP2K1). To prevent expression of the mutant alleles we introduced a LoxP flanked gene trap (GT) into the introns preceding the mutant exons, rendering the mutant alleles inactive. In the presence of Cre recombinase, the LoxP sites recombine, removing the GTs and allowing expression of the mutant MAP2K1 or GNAQ allele. By using a tamoxifen inducible version of Cre recombinase (CreERT2) that is specifically expressed in ECs (Cdh5CreERT2) we can: (1) activate the mutant alleles specifically in ECs and (2) by using low concentrations of tamoxifen activate the mutant alleles in a limited number of ECs.
RESULTS: CRISPR/Cas9 gene editing yielded one correctly targeted founder mouse for both MAP2K1 and GNAQ. Both founders gave germline transmission. Mice homozygous for the gene trapped MAP2K1-K57N allele were embryonic lethal, while mice homozygous for the gene trapped GNAQ-R183Q allele were growth retarded and featured neurological issues. Both phenotypes reprised the phenotype observed in conventional MAP2K1 and GNAQ knock-outs confirming the functionality of the GTs. MAP2K1-GT-K57N+/-;Cdh5-CreERT2+/- and GNAQ-GT-R183Q+/-;Cdh5-CreERT2+/- animals were generated and injected with tamoxifen. PCR on DNA extracted from these animals confirmed the removal of the GTs. Mutant transcript specific ddPCR confirmed expression of MAP2K1-K57N and GNAQ-R183Q. In a preliminary experiment we observed the occurrence of CM like lesions in the lungs of tamoxifen injected Gnaq-GT-R183Q+/-;Cdh5-CreERT2+/- mice.
CONCLUSION:
We have generated MAP2K1-GT-K57N+/-;Cdh5-CreERT2+/- and GNAQ-GT-R183Q+/-;Cdh5-CreERT2+/- mice and shown that we can activate expression of mutant MAP2K1 and GNAQ protein in endothelial cells. If tamoxifen injected animals develop AVMs or CMs, we can use our mouse models to test drugs as potential AVM and CM treatments.


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