Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a , Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.

Grainyhead-like genes are part of a highly conserved gene family that play a number of roles in ectoderm development and maintenance in mammals. Here we identify a novel allele of Grhl2, cleft-face 3 (clft3), in a mouse line recovered from an ENU mutagenesis screen for organogenesis defects. Homozygous clft3 mutants have a number of phenotypes in common with other alleles of Grhl2. We note a significant effect of genetic background on the clft3 phenotype. One of these is a reduction in size of the telencephalon where we find abnormal patterns of neural progenitor mitosis and apoptosis in mutant brains. Interestingly, Grhl2 is not expressed in the developing forebrain, suggesting this is a survival factor for neural progenitors exerting a paracrine effect on the neural tissue from the overlying ectoderm where Grhl2 is highly expressed.

rAAVrh74.MCK.GALGT2 is a surrogate gene therapy that inhibits muscular dystrophy in multiple animal models. Here, we report on a dose-response study of functional muscle GALGT2 expression as well as toxicity and biodistribution studies after systemic intravenous (i.v.) delivery of rAAVrh74.MCK.GALGT2. A dose of 4.3 × 1014vg/kg (measured with linear DNA standard) resulted in GALGT2-induced glycosylation in the majority of skeletal myofibers throughout the body and in almost all cardiomyocytes, while several lower doses also showed significant muscle glycosylation. No adverse clinical signs or treatment-dependent changes in tissue or organ pathology were noted at 1 or 3 months post-treatment. Blood cell and serum enzyme chemistry measures in treated mice were all within the normal range except for alkaline phosphatase (ALP) activity, which was elevated in serum but not in tissues. Some anti-rAAVrh74 capsid T cell responses were noted at 4 weeks post-treatment, but all such responses were not present at 12 weeks. Using intramuscular delivery, GALGT2-induced muscle glycosylation was increased in Cmah-deficient mice, which have a humanized sialoglycome, relative to wild-type mice, suggesting that use of mice may underestimate GALGT2 activity in human muscle. These data demonstrate safety and high transduction of muscles throughout the body plan with i.v. delivery of rAAVrh74.MCK.GALGT2.

KEY WORDSTubulin human forebrain development ABSTRACT Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations results from de novo heterozygous mutations in tubulin genes. However, the absolute requirement for most of these genes in development and disease has not been previously tested in genetic, loss of function models. Here we present two novel pathogenic tubulin alleles: a human TUBA1A missense variant with a phenotype more severe than most tubulinopathies and a mouse ENU allele of Tuba1a. Furthermore, we directly test the requirement for Tuba1a, Tuba8, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tuba8, Tubb2a or Tubb2b does not lead to cortical malformation phenotypes or impair survival. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. Thus, despite their functional similarity, the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.

Frizzled 2 (FZD2) is a transmembrane Wnt receptor. We previously identified a pathogenic human FZD2 variant in individuals with FZD2-associated autosomal dominant Robinow syndrome. The variant encoded a protein with a premature stop and loss of 17 amino acids, including a region of the consensus dishevelled-binding sequence. To model this variant, we used zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate a mouse allelic series. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with patient phenotypes. We also generated two germline mouse alleles with small deletions: Fzd2D3 and Fzd2D4. Homozygotes for each allele exhibit a highly penetrant cleft palate phenotype, shortened limbs compared with wild type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical Wnt signaling. In utero treatment with IIIC3a (a DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach for further investigating the mechanism of FZD2 phenotypes and demonstrates the utility of CRISPR knock-in mice as a tool for investigating the pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.