Hyo Min Park

Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9)-based therapeutics, especially those that can correct gene mutations via homology-directed repair, have the potential to revolutionize the treatment of genetic diseases. However, it is challenging to develop homology-directed repair-based therapeutics because they require the simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.Gold nanoparticles carrying Cas9 ribonucleoprotein and donor DNA, and complexed with endosomal disruptive polymers, correct the DNA mutation that causes Duchenne muscular dystrophy in mice, with minimal off-target effects.

Real-Time Noninvasive Imaging of Fatty Acid Uptake in Vivo

Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin sensitivity. To enable noninvasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic metabolic processes and pathological alterations in multiple disease models.

Specific bile acids inhibit hepatic fatty acid uptake in mice

Bile acids are known to play important roles as detergents in the absorption of hydrophobic nutrients and as signaling molecules in the regulation of metabolism. We tested the novel hypothesis that naturally occurring bile acids interfere with protein‐mediated hepatic long chain free fatty acid (LCFA) uptake. To this end, stable cell lines expressing fatty acid transporters as well as primary hepatocytes from mouse and human livers were incubated with primary and secondary bile acids to determine their effects on LCFA uptake rates. We identified ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) as the two most potent inhibitors of the liver‐specific fatty acid transport protein 5 (FATP5). Both UDCA and DCA were able to inhibit LCFA uptake by primary hepatocytes in a FATP5‐dependent manner. Subsequently, mice were treated with these secondary bile acids in vivo to assess their ability to inhibit diet‐induced hepatic triglyceride accumulation. Administration of DCA in vivo via injection or as part of a high‐fat diet significantly inhibited hepatic fatty acid uptake and reduced liver triglycerides by more than 50%. Conclusion: The data demonstrate a novel role for specific bile acids, and the secondary bile acid DCA in particular, in the regulation of hepatic LCFA uptake. The results illuminate a previously unappreciated means by which specific bile acids, such as UDCA and DCA, can impact hepatic triglyceride metabolism and may lead to novel approaches to combat obesity‐associated fatty liver disease. (HEPATOLOGY 2012)

In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease

Significance Like all essential metals in mammals, deficiency or excess of copper can be detrimental to health. We present a bioluminescent reporter based on copper-dependent uncaging of a d-luciferin substrate for selective, sensitive, and tissue-specific longitudinal imaging of labile copper pools in animal model systems. Application of this technology to monitor a diet-induced mouse model of nonalcoholic fatty liver disease, a disorder affecting ca. 100 million Americans, reveals hepatic copper deficiency and altered expression levels of copper homeostatic proteins that accompany glucose intolerance and weight gain. The results demonstrate the viability of this molecular imaging approach and connect copper dysregulation to metabolic liver disease, providing a platform for designing reactivity-based reporters for cell- and tissue-specific in vivo metal imaging. Copper is a required metal nutrient for life, but global or local alterations in its homeostasis are linked to diseases spanning genetic and metabolic disorders to cancer and neurodegeneration. Technologies that enable longitudinal in vivo monitoring of dynamic copper pools can help meet the need to study the complex interplay between copper status, health, and disease in the same living organism over time. Here, we present the synthesis, characterization, and in vivo imaging applications of Copper-Caged Luciferin-1 (CCL-1), a bioluminescent reporter for tissue-specific copper visualization in living animals. CCL-1 uses a selective copper(I)-dependent oxidative cleavage reaction to release d-luciferin for subsequent bioluminescent reaction with firefly luciferase. The probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of nonalcoholic fatty liver disease reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. The data connect copper dysregulation to metabolic liver disease and provide a starting point for expanding the toolbox of reactivity-based chemical reporters for cell- and tissue-specific in vivo imaging.

Differential requirement for de novo lipogenesis in cholangiocarcinoma and hepatocellular carcinoma of mice and humans.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most prevalent types of primary liver cancer. These malignancies have limited treatment options, resulting in poor patient outcomes. Metabolism reprogramming, including increased de novo lipogenesis, is one of the hallmarks of cancer. Fatty acid synthase (FASN) catalyzes the de novo synthesis of long‐chain fatty acids from acetyl‐coenzyme A and malonyl‐coenzyme A. Increased FASN expression has been reported in multiple tumor types, and inhibition of FASN expression has been shown to have tumor‐suppressing activity. Intriguingly, we found that while FASN is up‐regulated in human HCC samples, its expression is frequently low in human ICC specimens. Similar results were observed in mouse ICC models induced by different oncogenes. Ablating FASN in the mouse liver did not affect activated AKT and Notch (AKT/Notch intracellular domain 1) induced ICC formation in vivo. Furthermore, while both HCC and ICC lesions develop in mice following hydrodynamic injection of AKT and neuroblastoma Ras viral oncogene homolog oncogenes (AKT/Ras), deletion of FASN in AKT/Ras mice triggered the development almost exclusively of ICCs. In the absence of FASN, ICC cells might receive lipids for membrane synthesis through exogenous fatty acid uptake. In accordance with the latter hypothesis, ICC cells displayed high expression of fatty acid uptake‐related proteins and robust long‐chain fatty acid uptake. Conclusion: Our data demonstrate that FASN dependence is not a universal feature of liver tumors: while HCC development is highly dependent of FASN and its mediated lipogenesis, ICC tumorigenesis can be insensitive to FASN deprivation; our study supports novel therapeutic approaches to treat this pernicious tumor type with the inhibition of exogenous fatty acid uptake. (Hepatology 2016;63:1900‐1913)

Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours

Technologies that can safely edit genes in the brains of adult animals may revolutionize the treatment of neurological diseases and the understanding of brain function. Here, we demonstrate that intracranial injection of CRISPR–Gold, a nonviral delivery vehicle for the CRISPR–Cas9 ribonucleoprotein, can edit genes in the brains of adult mice in multiple mouse models. CRISPR–Gold can deliver both Cas9 and Cpf1 ribonucleoproteins, and can edit all of the major cell types in the brain, including neurons, astrocytes and microglia, with undetectable levels of toxicity at the doses used. We also show that CRISPR–Gold designed to target the metabotropic glutamate receptor 5 (mGluR5) gene can efficiently reduce local mGluR5 levels in the striatum after an intracranial injection. The effect can also rescue mice from the exaggerated repetitive behaviours caused by fragile X syndrome, a common single-gene form of autism spectrum disorders. CRISPR–Gold may significantly accelerate the development of brain-targeted therapeutics and enable the rapid development of focal brain-knockout animal models.Gene editing of a single gene in the brain of an adult mouse model of fragile X syndrome, achieved via the intracranial injection of a nonviral Cas9 delivery vehicle, rescues mice from the exaggerated repetitive behaviours caused by the disease.

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection

Significance Iron is a required metal nutrient for life, and its altered homeostasis is associated with a number of diseases. We present a bioluminescent reporter for visualizing iron pools in living animals, where iron-dependent uncaging of d-aminoluciferin enables sensitive and selective imaging of ferrous over ferric forms of iron in luciferase-expressing cell and mouse models. Application of this technology to a model of systemic bacterial infection reveals elevation of iron in infected tissues that accompany markers for increased iron acquisition and retention. These data establish the ability to assess iron status in living animals and provide a unique platform for studying its contributions to stages of health, aging, and disease. Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.

Measurement of long-chain fatty acid uptake into adipocytes

The ability of white and brown adipose tissue to efficiently take up long-chain fatty acids is key to their physiological functions in energy storage and thermogenesis, respectively. Several approaches have been taken to determine uptake rates by cultured cells and primary adipocytes including radio- and fluorescently labeled fatty acids. In addition, the recent description of activatable bioluminescent fatty acids has opened the possibility for expanding these in vitro approaches to real-time monitoring of fatty acid uptake kinetics by adipose depots in vivo. Here, we will describe some of the most useful experimental paradigms to quantitatively determine long-chain fatty acid uptake by adipocytes in vitro and provide the reader with detailed instruction on how bioluminescent probes for in vivo imaging can be synthesized and used in living mice.

A Biocompatible “Split Luciferin” Reaction and Its Application for Non‐Invasive Bioluminescent Imaging of Protease Activity in Living Animals

The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real‐time non‐invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the “split luciferin” biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide‐specific substrates. In this unit, we describe several applications of this “split luciferin” reaction for quantification of protease activities in test tube assays and living animals. Curr. Protoc. Chem. Biol. 6:169‐189

A System for In Vivo Imaging of Hepatic Free Fatty Acid Uptake.

Alterations in hepatic free fatty acid (FFA) uptake and metabolism contribute to the development of prevalent liver disorders such as hepatosteatosis. However, detecting dynamic changes in FFA uptake by the liver in live model organisms has proven difficult. To enable noninvasive real-time imaging of FFA flux in the liver, we generated transgenic mice with liver-specific expression of luciferase and performed bioluminescence imaging with an FFA probe. Our approach enabled us to observe the changes in FFA hepatic uptake under different physiological conditions in live animals. By using this method, we detected a decrease in FFA accumulation in the liver after mice were given injections of deoxycholic acid and an increase after they were fed fenofibrate. In addition, we observed diurnal regulation of FFA hepatic uptake in living mice. Our imaging system appears to be a useful and reliable tool for studying the dynamic changes in hepatic FFA flux in models of liver disease.