Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Natural genetic variation in a dopamine receptor is associated with variation in female fertility in Drosophila melanogaster.

Fertility is a major component of fitness but its genetic architecture remains poorly understood. Using a full diallel cross of 50 Drosophila Genetic Reference Panel inbred lines with whole genome sequences, we found substantial genetic variation in fertility largely attributable to females. We mapped genes associated with variation in female fertility by genome-wide association analysis of common variants in the fly genome. Validation of candidate genes by RNAi knockdown confirmed the role of the dopamine 2-like receptor (Dop2R) in promoting egg laying. We replicated the Dop2R effect in an independently collected productivity dataset and showed that the effect of the Dop2R variant was mediated in part by regulatory gene expression variation. This study demonstrates the strong potential of genome-wide association analysis in this diverse panel of inbred strains and subsequent functional analyses for understanding the genetic architecture of fitness traits.

A Cyclin E Centered Genetic Network Contributes to Alcohol-Induced Variation in Drosophila Development.

Prenatal exposure to ethanol causes a wide range of adverse physiological, behavioral and cognitive consequences. However, identifying allelic variants and genetic networks associated with variation in susceptibility to prenatal alcohol exposure is challenging in human populations, since time and frequency of exposure and effective dose cannot be determined quantitatively and phenotypic manifestations are diverse. Here, we harnessed the power of natural variation in the Drosophila melanogaster Genetic Reference Panel (DGRP) to identify genes and genetic networks associated with variation in sensitivity to developmental alcohol exposure. We measured development time from egg to adult and viability of 201 DGRP lines reared on regular or ethanol- supplemented medium and identified polymorphisms associated with variation in susceptibility to developmental ethanol exposure. We also documented genotype-dependent variation in sensorimotor behavior after developmental exposure to ethanol using the startle response assay in a subset of 39 DGRP lines. Genes associated with development, including development of the nervous system, featured prominently among genes that harbored variants associated with differential sensitivity to developmental ethanol exposure. Many of them have human orthologs and mutational analyses and RNAi targeting functionally validated a high percentage of candidate genes. Analysis of genetic interaction networks identified Cyclin E (CycE) as a central, highly interconnected hub gene. Cyclin E encodes a protein kinase associated with cell cycle regulation and is prominently expressed in ovaries. Thus, exposure to ethanol during development of Drosophila melanogaster might serve as a genetic model for translational studies on fetal alcohol spectrum disorder.

Genetic architecture of natural variation in visual senescence in Drosophila.

Senescence, i.e., functional decline with age, is a major determinant of health span in a rapidly aging population, but the genetic basis of interindividual variation in senescence remains largely unknown. Visual decline and age-related eye disorders are common manifestations of senescence, but disentangling age-dependent visual decline in human populations is challenging due to inability to control genetic background and variation in histories of environmental exposures. We assessed the genetic basis of natural variation in visual senescence by measuring age-dependent decline in phototaxis using Drosophila melanogaster as a genetic model system. We quantified phototaxis at 1, 2, and 4 wk of age in the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and found an average decline in phototaxis with age. We observed significant genetic variation for phototaxis at each age and significant genetic variation in senescence of phototaxis that is only partly correlated with phototaxis. Genome-wide association analyses in the DGRP and a DGRP-derived outbred, advanced intercross population identified candidate genes and genetic networks associated with eye and nervous system development and function, including seven genes with human orthologs previously associated with eye diseases. Ninety percent of candidate genes were functionally validated with targeted RNAi-mediated suppression of gene expression. Absence of candidate genes previously implicated with longevity indicates physiological systems may undergo senescence independent of organismal life span. Furthermore, we show that genes that shape early developmental processes also contribute to senescence, demonstrating that senescence is part of a genetic continuum that acts throughout the life span.

Spontaneous mutations and the origin and maintenance of quantitative genetic variation.

Mutation and natural selection shape the genetic variation in natural populations. Here, we directly estimated the spontaneous mutation rate by sequencing new Drosophila mutation accumulation lines maintained with minimal natural selection. We inferred strong stabilizing natural selection on quantitative traits because genetic variation among wild-derived inbred lines was much lower than predicted from a neutral model and the mutational effects were much larger than allelic effects of standing polymorphisms. Stabilizing selection could act directly on the traits, or indirectly from pleiotropic effects on fitness. However, our data are not consistent with simple models of mutation-stabilizing selection balance; therefore, further empirical work is needed to assess the balance of evolutionary forces responsible for quantitative genetic variation.