A porcine model of Fanconi anemia

Abstract

Although small animal models of Fanconi anemia (FA) are useful, they do not faithfully replicate many of the clinical features seen in FA patients. We reasoned that a porcine model of FA with its similar physiology and a relatively long lifespan would produce a phenotype more similar to human FA. Targeting FANCA in domestic swine resulted in skeletal abnormalities and extreme sensitivity to interstrand DNA cross-linking agents. In addition, FANCA disruption followed by mitomycin C treatment resulted in a > 10-fold increase in chromosomal radials, a finding that is considered diagnostic for human FA. Bone marrow derived, hematopoietic progenitor cells from a FANCA null pig showed a 75% reduction in colony forming activity compared to wild type. Evaluation of steady state hematopoiesis in the peripheral blood revealed the gradual development of red cell macrocytosis and a reduction in circulating neutrophils. Targeting of FANCD2 failed to produce any biallelic animals demonstrating the loss of FANCD2 function is embryonic lethal in pigs. These results indicate that a porcine model of FANCA holds promise for the development of strategies to prevent the development of bone marrow failure and malignancies in patients with FA.

Fig 1. In vitro targeting of FANCA exon 4 in primary pig fibroblasts.

(A) Fetal pig fibroblasts were nucleofected with CRISPR-Cas9 targeting FANCA exon 4 using sgRNA and a GFP marker. After 2 days of culture, GFP+ cells were cell-sorted into a 96-well plate expanded for 1 week and then genotyped for FANCA exon 4 mutations. Three clones were selected for phenotypic analysis and tested for their sensitivity to DEB in colony forming assay; Control ^(WT/+1), Mutant A^(+1/+2) and Mutant B^(+250/+220). Both mutant clones showed clear hypersensitivity to DEB consistent with loss of FANCA function.

Fig 2. Creation of FANCA KO pigs using CRISPR-Cas9 targeting.

(A) Pig oocytes were targeted for FANCA by microinjection of sgRNA and mRNA encoding Cas9 followed by in vitro fertilization. Zygotes were cultured to the blastocyst/morula stage then transferred to a surrogate mother. Initial progeny underwent genotypic and phenotypic analysis. As mosaicism often occurs, heterozygous animals were bred creating an F1 generation with biallelic out-of-frame FANCA pigs. (B) Schematic of Exon 4 targeting construct and examples of the resulting indel events.

Fig 3. Polydactyl dew claws in a FANCA exon 4 targeted piglet.

A) Image of front limbs showing extra dewclaws of variable size on the medial aspect of both front limbs of F1 generation piglet 67−2. B) X-rays of 67−2 showing an extra dew claws on (white arrows) both front limbs. C) Anatomy of normal porcine foot.

Fig 4. FANCA-/- pig tongue histology is abnormal.

Mutant pig (A,B) and wild type (C,D) tongue sections were stained with H&E or labeled with Ki-67 to visualize proliferation. Arrows indicate areas of delayed differentiation. Total magnification 100x.

Fig 5. Evaluation of sensitivity to DNA cross linking in primary cells from a FANCA biallelic exon 4 targeted pig.

A) A colony forming assay was performed on primary fibroblasts from the biallelic piglet 67−2. Cells were plated in duplicate in 6 well plates at the shown concentrations of DEB. A wild-type littermate (67−4) and the transformed human cell line GM639 were used as wild-type controls. The transformed human cell line GM6914 was included as a FANCA-deficient control. Combined data showing total colony counts from 3 independent experiments is shown. Error bars indicate SEM.

Fig 6. MMC exposure results in chromosomal aberrations in a biallelic FANCA exon 4 targeted pig.

WT and 67−2 ^{(+7, −2)} peripheral blood mononuclear cells were cultured in the presence of PHA, treated with 20 ng/ml of MMC followed by colcemid arrest and metaphases were harvested for cytogenetic analysis. A) Quantification of aberrations/cell in wild type and FANCA KO pig lymphocytes exposed to either 0 or 20 ng/ml MMC treatment for 48h. B) Percent of cells with radials in FANCA KO and WT pig lymphocytes treated with 0 or 20 ng/ml MMC. CD) Examples of chromosomal aberrations and radials, as indicated by arrows, in metaphase spreads from FANCA KO pig lymphocytes post MMC treatment. (E) The frequency of colony formation by cultured tail fibroblasts derived from a control pig and the 67−2 FANCA^-/- (FA-/-) pig grown in the presence or absence of 12nM MMC. Fibroblasts from 67−2 were also transduced with a lentivirus expressing human FANCA (FA-/- corrected) prior to MMC treatment. Mean and SEM is indicated (p value determined using a student’s t-test.

Fig 7. FANCA^-/- pigs exhibit hematopoietic defects.

A) CBC profiling at multiple time points shows progressive defects in multiple lineages. B) Colony forming cell (CFC) frequency in cryopreserved bone marrow is significantly lower in mutant animals than in normal controls. The mean and standard error of the mean from triplicate plates in a representative CFC assay is shown. Student’s t-test P value is indicated. EOS, Eosinophils; HCT, hematocrit; HGB, hemoglobin; LYMPH, lymphocytes; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MONO, monocytes; NEUT, neutrophils; PLT, platelets; RBC, red blood cell; WBC, white blood cell. Bold indicates outside of normal range. * = High value, † = Low value.

Fig 8. Sensitivity of mosaic piglet (126−3) to DNA cross-linking agents.

Colony forming activity for fibroblasts from the wild type and mutant human and pig cells following exposure to increasing concentrations of the DNA cross-linking agent DEB is shown.