Liver directed adeno-associated viral vectors to treat metabolic disease.

The liver is the metabolic center of the body and an ideal target for gene therapy of inherited metabolic disorders (IMDs). Adeno-associated viral (AAV) vectors can deliver transgenes to the liver with high efficiency and specificity and a favorable safety profile. Recombinant AAV vectors contain only the transgene cassette, and their payload is converted to non-integrating circular double-stranded DNA episomes, which can provide stable expression from months to years. Insights from cellular studies and preclinical animal models have provided valuable information about AAV capsid serotypes with a high liver tropism. These vectors have been applied successfully in the clinic, particularly in trials for hemophilia, resulting in the first approved liver-directed gene therapy. Lessons from ongoing clinical trials have identified key factors affecting efficacy and safety that were not readily apparent in animal models. Circumventing pre-existing neutralizing antibodies to the AAV capsid, and mitigating adaptive immune responses to transduced cells are critical to achieving therapeutic benefit. Combining the high efficiency of AAV delivery with genome editing is a promising path to achieve more precise control of gene expression. The primary safety concern for liver gene therapy with AAV continues to be the small risk of tumorigenesis from rare vector integrations. Hepatotoxicity is a key consideration in the safety of neuromuscular gene therapies which are applied at substantially higher doses. The current knowledge base and toolkit for AAV is well developed, and poised to correct some of the most severe IMDs with liver-directed gene therapy.

In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene.

Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested "Repair Drive", a novel method for improving targeted gene insertion in the liver by selectively expanding correctly repaired hepatocytes in vivo. Our system consists of transient conditioning of the liver by knocking down an essential gene, and delivery of an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene. Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing.

LPA disruption with AAV-CRISPR potently lowers plasma apo(a) in transgenic mouse model: A proof-of-concept study.

Lipoprotein(a) (Lp(a)) represents a unique subclass of circulating lipoprotein particles and consists of an apolipoprotein(a) (apo(a)) molecule covalently bound to apolipoprotein B-100. The metabolism of Lp(a) particles is distinct from that of low-density lipoprotein (LDL) cholesterol, and currently approved lipid-lowering drugs do not provide substantial reductions in Lp(a), a causal risk factor for cardiovascular disease. Somatic genome editing has the potential to be a one-time therapy for individuals with extremely high Lp(a). We generated an LPA transgenic mouse model expressing apo(a) of physiologically relevant size. Adeno-associated virus (AAV) vector delivery of CRISPR-Cas9 was used to disrupt the LPA transgene in the liver. AAV-CRISPR nearly completely eliminated apo(a) from the circulation within a week. We performed genome-wide off-target assays to determine the specificity of CRISPR-Cas9 editing within the context of the human genome. Interestingly, we identified intrachromosomal rearrangements within the LPA cDNA in the transgenic mice as well as in the LPA gene in HEK293T cells, due to the repetitive sequences within LPA itself and neighboring pseudogenes. This proof-of-concept study establishes the feasibility of using CRISPR-Cas9 to disrupt LPA in vivo, and highlights the importance of examining the diverse consequences of CRISPR cutting within repetitive loci and in the genome globally.

Intestinal Deletion of 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Promotes Expansion of the Resident Stem Cell Compartment.

BACKGROUND: The intestine occupies the critical interface between cholesterol absorption and excretion. Surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. Here, we investigate the physiological importance of this pathway through genetic deletion of the rate-limiting enzyme. METHODS: Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) in intestinal villus and crypt epithelial cells were generated using a Villin-Cre transgene. Plasma lipids, intestinal morphology, mevalonate pathway metabolites, and gene expression were analyzed. RESULTS: Mice with intestine-specific loss of Hmgcr were markedly smaller at birth, but gain weight at a rate similar to wild-type littermates, and are viable and fertile into adulthood. Intestine lengths and weights were greater relative to body weight in both male and female Hmgcr intestinal knockout mice. Male intestinal knockout had decreased plasma cholesterol levels, whereas fasting triglycerides were lower in both sexes. Lipidomics revealed substantial reductions in numerous nonsterol isoprenoids and sterol intermediates within the epithelial layer, but cholesterol levels were preserved. Hmgcr intestinal knockout mice also showed robust activation of SREBP-2 (sterol-regulatory element binding protein-2) target genes in the epithelium, including the LDLR (low-density lipoprotein receptor). At the cellular level, loss of Hmgcr is compensated for quickly after birth through a dramatic expansion of the stem cell compartment, which persists into adulthood. CONCLUSIONS: Loss of Hmgcr in the intestine is compatible with life through compensatory increases in intestinal absorptive surface area, LDLR expression, and expansion of the resident stem cell compartment.

Nanoparticle mediated increased insulin-like growth factor 1 expression enhances human placenta syncytium function.

INTRODUCTION: Placental dysfunction is an underlying cause of many major obstetric diseases and treatment options for complications like fetal growth restriction (FGR) are limited .We previously demonstrated nanoparticle delivery of the human insulin-like growth factor 1 (hIGF1) transgene under control of the trophoblast-specific PLAC1 promoter maintains normal fetal growth in a surgically-induced FGR mouse model. However, uptake by human placental syncytiotrophoblast has yet to be determined. METHODS: An ex vivo human placenta perfusion model, term placenta villous fragments, and other in vitro syncytiotrophoblast models were used to determine nanoparticle uptake, transgene expression, and functional responses under oxidative stress conditions. RESULTS: In the ex vivo perfusion, fluorescence from a Texas-Red conjugated nanoparticle increased in maternal perfusate upon nanoparticle addition and declined by the conclusion of the experiment (P < 0.001. Fluorescent histology confirmed localization in the syncytiotrophoblasts. No Texas-Red fluorescence was detected in the fetal perfusate. Transgene expression of hIGF1 in differentiated BeWo cells, isolated primary trophoblasts and fragments was increased compared to untreated (55,000-fold, P = 0.0003; 95-fold, P = 0.003; 400-fold, P < 0.001, respectively). Functionally, increased hIGF1 expression in villous fragments resulted in translocation of glucose transporter 1 to the syncytiotrophoblast cell membrane and under conditions of oxidative stress in BeWo cells, protected against increased cell death (P < 0.01) and decreased mitochondrial activity (P < 0.01). CONCLUSION: The current study confirms that our nanoparticle is capable of uptake in human placental syncytium which results in enhanced transgene expression, functional changes to cellular activity and protection against increased oxidative stress.

Optical tissue clearing in combination with perfusion and immunofluorescence for placental vascular imaging.

Imaging of placental tissues is a difficult task, because of specific for this organ complex multicellular and 3D tissue structure. The tissue clearing systems (X-CLARITY) system is a valuable tool for the examining the expression of molecular pathways in whole tissues and organs, originally developed for brain imaging.In the present report, we utilized this technology for the examination of placental vasculature and protein expression in perfused human placental tissue.The placental tissue was sufficiently cleared with preservation of endothelial staining and fluorescent markers, allowing visualization using confocal microscopy. The CLARITY method and X-CLARITY system is a valuable tool in placental imaging.

Effect of maternal high-fat diet on key components of the placental and hepatic endocannabinoid system.

Maternal obesity in pregnancy has been linked to a spectrum of adverse developmental changes. Involvement of eCBs in obesity is well characterized. However, information regarding eCB physiology in obesity associated with pregnancy is sparse. This study evaluated fetomaternal hepatic, systemic, and placental eCB molecular changes in response to maternal consumption of a HFD. From ≥9 mo before conception, nonpregnant baboons ( Papio spp.) were fed a diet of either 45 (HFD; n = 11) or 12% fat or a control diet (CTR; n = 11), and dietary intervention continued through pregnancy. Maternal and fetal venous plasma samples were evaluated using liquid chromatography-mass spectrometry to quantify AEA and 2-AG. Placental, maternal and fetal hepatic tissues were analyzed using RT-PCR, Western blot, and immunohistochemistry. mRNA and protein expression of endocannabinoid receptors (CB1R and CB2R), FAAH, DAGL, MAGL, and COX-2 were determined. Statistical analyses were performed with the nonparametric Scheirer-Ray-Hare extension of the Kruskal-Wallis test to analyze the effects of diet (HFD vs. CTR), fetal sex (male vs. female), and the diet × sex interaction. Fetal weight was influenced by fetal sex but not by maternal diet. The increase in maternal weight in animals fed the HFD vs. the CTR diet approached significance ( P = 0.055). Maternal circulating 2-AG concentrations increased, and fetal circulating concentrations decreased in the HFD group, independently of fetal sex. CB1R receptor expression was detected in syncytiotrophoblasts (HFD) and the fetal endothelium (CTR and HFD). Placental CB2R protein expression was higher in males and lower in female fetuses in the HFD group. Fetal hepatic CB2R, FAAH, COX-2 (for both fetal sexes), and DAGLα (in male fetuses) protein expression decreased in the HFD group compared with the CTR group. We conclude that consumption of a HFD during pregnancy results in fetal systemic 2-AG and hepatic eCB deficiency.