Targeting AAV vectors to the central nervous system by engineering capsid-receptor interactions that enable crossing of the blood-brain barrier.

Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated virus (AAV) capsids that transduce the brain through the introduction of de novo interactions with 2 proteins expressed on the mouse blood-brain barrier (BBB), LY6A or LY6C1. The in vivo tropisms of these capsids are predictable as they are dependent on the cell- and strain-specific expression of their target protein. This approach generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms within a single round of screening in vitro and secondary validation in vivo thereby reducing the use of animals in comparison to conventional multi-round in vivo selections. The reproducible and quantitative data derived via this method enabled both saturation mutagenesis and machine learning (ML)-guided exploration of the capsid sequence space. Notably, during our validation process, we determined that nearly all published AAV capsids that were selected for their ability to cross the BBB in mice leverage either the LY6A or LY6C1 protein, which are not present in primates. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.

AAV5 delivery of CRISPR-Cas9 supports effective genome editing in mouse lung airway.

Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. The NIH Somatic Cell Genome Editing (SCGE) Consortium is developing safe and effective methods for genome editing in disease tissues. Methods developed by consortium members are independently validated by the SCGE small animal testing center to establish rigor and reproducibility. We have developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. After intratracheal injection of the AAV serotype 5 (AAV5)-packaged S. pyogenes Cas9 (SpCas9) and single guide RNAs (sgRNAs), we observed ∼19%-26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. This highly effective AAV delivery platform will facilitate the study of therapeutic genome editing in the lung and other tissue types.

Susceptibility of Rat Steatotic Liver to Ischemia-Reperfusion Is Treatable With Liver-Selective Matrix Metalloproteinase Inhibition.

BACKGROUND AND AIMS: This study examined whether enhanced susceptibility of steatotic liver to ischemia-reperfusion (I/R) injury is due to impaired recruitment of bone marrow (BM) progenitors of liver sinusoidal endothelial cells (LSECs, also called sinusoidal endothelial cell progenitor cells [sprocs]) with diminished repair of injured LSECs and whether restoring signaling to recruit BM sprocs reduces I/R injury. APPROACH AND RESULTS: Hepatic vessels were clamped for 1 hour in rats fed a high-fat, high-fructose (HFHF) diet for 5, 10, or 15 weeks. Matrix metalloproteinase 9 (MMP-9) antisense oligonucleotides (ASO) or an MMP inhibitor were used to induce liver-selective MMP-9 inhibition. HFHF rats had mild, moderate, and severe steatosis, respectively, at 5, 10, and 15 weeks. I/R injury was enhanced in HFHF rats; this was accompanied by complete absence of hepatic vascular endothelial growth factor (VEGF)-stromal cell-derived factor 1 (sdf1) signaling, leading to lack of BM sproc recruitment. Liver-selective MMP-9 inhibition to protect against proteolytic cleavage of hepatic VEGF using either MMP-9 ASO or intraportal MMP inhibitor in 5-week and 10-week HFHF rats enhanced hepatic VEGF-sdf1 signaling, increased BM sproc recruitment, and reduced alanine aminotransferase (ALT) by 92% and 77% at 5 weeks and by 80% and 64% at 10 weeks of the HFHF diet, respectively. After I/R injury in 15-week HFHF rats, the MMP inhibitor reduced active MMP-9 expression by 97%, ameliorated histologic evidence of injury, and reduced ALT by 58%, which is comparable to control rats sustaining I/R injury. Rescue therapy with intraportal MMP inhibitor, given after ischemia, in the 5-week HFHF rat reduced ALT by 71% and reduced necrosis. CONCLUSIONS: Lack of signaling to recruit BM sprocs that repair injured LSECs renders steatotic liver more susceptible to I/R injury. Liver-selective MMP-9 inhibition enhances VEGF-sdf1 signaling and recruitment of BM sprocs, which markedly protects against I/R injury, even in severely steatotic rats.

The RNA polymerase III repressor MAF1 is regulated by ubiquitin-dependent proteasome degradation and modulates cancer drug resistance and apoptosis.

MAF1 homolog, negative regulator of RNA polymerase III (MAF1) is a key repressor of RNA polymerase (pol) III-dependent transcription and functions as a tumor suppressor. Its expression is frequently down-regulated in primary human hepatocellular carcinomas (HCCs). However, this reduction in MAF1 protein levels does not correlate with its transcript levels, indicating that MAF1 is regulated post-transcriptionally. Here, we demonstrate that MAF1 is a labile protein whose levels are regulated through the ubiquitin-dependent proteasome pathway. We found that MAF1 ubiquitination is enhanced upon mTOR complex 1 (TORC1)-mediated phosphorylation at Ser-75. Moreover, we observed that the E3 ubiquitin ligase cullin 2 (CUL2) critically regulates MAF1 ubiquitination and controls its stability and subsequent RNA pol III-dependent transcription. Analysis of the phenotypic consequences of modulating either CUL2 or MAF1 protein expression revealed changes in actin cytoskeleton reorganization and altered sensitivity to doxorubicin-induced apoptosis. Repression of RNA pol III-dependent transcription by chemical inhibition or knockdown of BRF1 RNA pol III transcription initiation factor subunit (BRF1) enhanced HCC cell sensitivity to doxorubicin, suggesting that MAF1 regulates doxorubicin resistance in HCC by controlling RNA pol III-dependent transcription. Together, our results identify the ubiquitin proteasome pathway and CUL2 as important regulators of MAF1 levels. They suggest that decreases in MAF1 protein underlie chemoresistance in HCC and perhaps other cancers and point to an important role for MAF1 and RNA pol III-mediated transcription in chemosensitivity and apoptosis.

Recent advances in genome editing for cardiovascular disease.

PURPOSE OF REVIEW: This review highlights recent progress in applying genome editing to the study and treatment of cardiovascular disease (CVD). RECENT FINDINGS: Recent work has shown that genome editing can be used to determine the pathogenicity of variants of unknown significance in patient-derived induced pluripotent stem cells. These cells can also be used to test therapeutic genome editing approaches in a personalized manner. Somatic genome editing holds great promise for the treatment of CVD, and important proof of concept experiments have already been performed in animal models. Here we briefly review recent progress in patient-derived cells, as well as the development of somatic genome-editing therapies for CVD, with a particular focus on liver and heart. SUMMARY: Translating this technology into the clinic will require precise editing enzymes, efficient delivery systems, and mitigation of off-target events and immune responses. Further development of these technologies will improve diagnostics and enable permanent correction of some of the most severe forms of CVD.

Neuronal deficiency of ARV1 causes an autosomal recessive epileptic encephalopathy.

We report an individual who presented with severe neurodevelopmental delay and an intractable infantile-onset seizure disorder. Exome sequencing identified a homozygous single nucleotide change that abolishes a splice donor site in the ARV1 gene (c.294 + 1G > A homozygous). This variant completely prevented splicing in minigene assays, and resulted in exon skipping and an in-frame deletion of 40 amino acids in primary human fibroblasts (NP_073623.1: p.(Lys59_Asn98del). The p.(Lys59_Asn98del) and previously reported p.(Gly189Arg) ARV1 variants were evaluated for protein expression and function. The p.(Gly189Arg) variant partially rescued the temperature-dependent growth defect in arv1Δ yeast, while p.(Lys59-Asn98del) completely failed to rescue at restrictive temperature. In contrast to wild type human ARV1, neither variant expressed detectable levels of protein in mammalian cells. Mice with a neuronal deletion of Arv1 recapitulated the human phenotype, exhibiting seizures and a severe survival defect in adulthood. Our data support ARV1 deficiency as a cause of autosomal recessive epileptic encephalopathy.

A Comprehensive Atlas of AAV Tropism in the Mouse.

Gene therapy with Adeno-Associated Viral (AAV) vectors requires knowledge of their tropism within the body. Here we analyze the tropism of ten naturally occurring AAV serotypes (AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10 and AAVrh74) following systemic delivery into male and female mice. A transgene expressing ZsGreen and Cre recombinase was used to identify transduction in a cell-dependent manner based on fluorescence. Cre-driven activation of tdTomato fluorescence offered superior sensitivity for transduced cells. All serotypes except AAV3B and AAV4 had high liver tropism. Fluorescence activation revealed transduction of unexpected tissues, including adrenals, testes and ovaries. Rare transduced cells within tissues were also readily visualized. Biodistribution of AAV genomes correlated with fluorescence, except in immune tissues. AAV4 was found to have a pan-endothelial tropism while also targeting pancreatic beta cells. This public resource enables selection of the best AAV serotypes for basic science and preclinical applications in mice.

Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine.

Control of volatile acidity (VA) is a major issue for wine quality. In this study, we investigated the production of VA by a deletion mutant of the fermentation stress response gene AAF1 in the budding yeast Saccharomyces cerevisiae. Fermentations were carried out in commercial Chardonnay grape must to mimic industrial wine-making conditions. We demonstrated that a wine yeast strain deleted for AAF1 reduced acetic acid levels in wine by up to 39.2% without increasing the acetaldehyde levels, revealing a potential for industrial application. Deletion of the cytosolic aldehyde dehydrogenase gene ALD6 also reduced acetic acid levels dramatically, but increased the acetaldehyde levels by 41.4%, which is not desired by the wine industry. By comparison, ALD4 and the AAF1 paralog RSF2 had no effects on acetic acid production in wine. Deletion of AAF1 was detrimental to the growth of ald6Δ and ald4Δald6Δ mutants, but had no effect on acetic acid production. Overexpression of AAF1 dramatically increased acetic acid levels in wine in an Ald6p-dependent manner, indicating that Aaf1p regulates acetic acid production mainly via Ald6p. Overexpression of AAF1 in an ald4Δald6Δ strain produced significantly more acetic acid in wine than the ald4Δald6Δ mutant, suggesting that Aaf1p may also regulate acetic acid synthesis independently of Ald4p and Ald6p.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.

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.

MAF1, a repressor of RNA polymerase III-dependent transcription, regulates bone mass.

MAF1, a key repressor of RNA polymerase (pol) III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show that MAF1 plays a critical role in regulating osteoblast differentiation and bone mass. Global deletion of MAF1 (Maf1-/- mice) produced a high bone mass phenotype. However, osteoblasts isolated from Maf1-/- mice showed reduced osteoblastogenesis ex vivo. Therefore, we determined the phenotype of mice overexpressing MAF1 in cells from the mesenchymal lineage (Prx1-Cre;LSL-MAF1 mice). These mice showed increased bone mass. Ex vivo, cells from these mice showed enhanced osteoblastogenesis concordant with their high bone mass phenotype. Thus, the high bone mass phenotype in Maf1-/- mice is likely due to confounding effects from the global absence of MAF1. MAF1 overexpression promoted osteoblast differentiation of ST2 cells while MAF1 downregulation inhibited differentiation, indicating MAF1 enhances osteoblast formation. However, other perturbations used to repress RNA pol III transcription, inhibited osteoblast differentiation. However, decreasing RNA pol III transcription through these perturbations enhanced adipogenesis in ST2 cells. RNA-seq analyzed the basis for these opposing actions on osteoblast differentiation. The different modalities used to perturb RNA pol III transcription resulted in distinct gene expression changes, indicating that this transcription process is highly sensitive and triggers diverse gene expression programs and phenotypic outcomes. Specifically, MAF1 induced genes known to promote osteoblast differentiation. Furthermore, genes that are induced during osteoblast differentiation displayed codon bias. Together, these results reveal a novel role for MAF1 and RNA pol III-mediated transcription in osteoblast fate determination, differentiation, and bone mass regulation.

The Saccharomyces cerevisiae fermentation stress response protein Igd1p/Yfr017p regulates glycogen levels by inhibiting the glycogen debranching enzyme.

Wine fermentation imposes a number of stresses on Saccharomyces cerevisiae, and wine yeasts respond to this harsh environment by altering their transcriptional profile (Marks et al., 2008). We have labeled this change in gene expression patterns the fermentation stress response (FSR). An important component of the FSR is the increased expression of 62 genes for which no function has been identified for their protein products. We hypothesize that a function for these proteins may only be revealed late in grape must fermentation, when the yeast cells are facing conditions much more extreme than those normally encountered in laboratory media. We used affinity copurification to identify interaction partners for the FSR protein Yfr017p, and found that it interacts specifically with the glycogen debranching enzyme (Gdb1p). The expression of both of these proteins is strongly induced during wine fermentation. Therefore, we investigated the role of Yfr017p in glycogen metabolism by constructing wine yeast strains that lack this protein. These YFR017C null cells displayed a significant reduction in their ability to accumulate glycogen during aerobic growth and fermentation. Moreover, Yfr017p inhibits Gdb1p activity in vitro. These results suggest that Yfr017p functions as an inhibitor of Gdb1p, enhancing the ability of yeast cells to store glucose as glycogen. Therefore, we propose IGD1 (for inhibitor of glycogen debranching) as a gene name for the YFR017C ORF.

Maf1 and Repression of RNA Polymerase III-Mediated Transcription Drive Adipocyte Differentiation.

RNA polymerase (pol) III transcribes a variety of small untranslated RNAs involved in transcription, RNA processing, and translation. RNA pol III and its components are altered in various human developmental disorders, yet their roles in cell fate determination and development are poorly understood. Here we demonstrate that Maf1, a transcriptional repressor, promotes induction of mouse embryonic stem cells (mESCs) into mesoderm. Reduced Maf1 expression in mESCs and preadipocytes impairs adipogenesis, while ectopic Maf1 expression in Maf1-deficient cells enhances differentiation. RNA pol III repression by chemical inhibition or knockdown of Brf1 promotes adipogenesis. Altered RNA pol III-dependent transcription produces select changes in mRNAs with a significant enrichment of adipogenic gene signatures. Furthermore, RNA pol III-mediated transcription positively regulates long non-coding RNA H19 and Wnt6 expression, established adipogenesis inhibitors. Together, these studies reveal an important and unexpected function for RNA pol III-mediated transcription and Maf1 in mesoderm induction and adipocyte differentiation.

Elevated TATA-binding protein expression drives vascular endothelial growth factor expression in colon cancer.

The TATA-binding protein (TBP) plays a central role in eukaryotic gene transcription. Given its key function in transcription initiation, TBP was initially thought to be an invariant protein. However, studies showed that TBP expression is upregulated by oncogenic signaling pathways. Furthermore, depending on the cell type, small increases in cellular TBP amounts can induce changes in cellular growth properties towards a transformed phenotype. Here we sought to identify the specific TBP-regulated gene targets that drive its ability to induce tumorigenesis. Using microarray analysis, our results reveal that increases in cellular TBP concentrations produce selective alterations in gene expression that include an enrichment for genes involved in angiogenesis. Accordingly, we find that TBP levels modulate VEGFA expression, the master regulator of angiogenesis. Increases in cellular TBP amounts induce VEGFA expression and secretion to enhance cell migration and tumor vascularization. TBP mediates changes in VEGFA transcription requiring its recruitment at a hypoxia-insensitive proximal TSS, revealing a mechanism for VEGF regulation under non-stress conditions. The results are clinically relevant as TBP expression is significantly increased in both colon adenocarcinomas as well as adenomas relative to normal tissue. Furthermore, TBP expression is positively correlated with VEGFA expression. Collectively, these studies support the idea that increases in TBP expression contribute to enhanced VEGFA transcription early in colorectal cancer development to drive tumorigenesis.

Enhancing the natural folate level in wine using bioengineering and stabilization strategies.

Folate deficiency is linked to many diseases, some of which may have higher probability in individuals with alcohol-induced alterations in one-carbon metabolism. Our study shows that folate content in commercial wine is not related to white or red varieties, but associated with the yeast that is used to produce the wine. The stability of folate in these wines, once opened for consumption, did not correlate with total phenolic or sulfite content. In addition, we employed yeast bioengineering to fortify wine with folate. We confirmed by overexpression that FOL2 was the key gene encoding the rate-limiting step of folate biosynthesis in wine yeast. In this study, we also show that overexpression of other folate biosynthesis genes, including ABZ1, ABZ2, DFR1, FOL1 and FOL3, had no effect on folate levels in wine. Ensuring stability of the increased natural folate in all wines was achieved by the addition of ascorbate.

Functional analyses of NSF1 in wine yeast using interconnected correlation clustering and molecular analyses.

Analyzing time-course expression data captured in microarray datasets is a complex undertaking as the vast and complex data space is represented by a relatively low number of samples as compared to thousands of available genes. Here, we developed the Interdependent Correlation Clustering (ICC) method to analyze relationships that exist among genes conditioned on the expression of a specific target gene in microarray data. Based on Correlation Clustering, the ICC method analyzes a large set of correlation values related to gene expression profiles extracted from given microarray datasets. ICC can be applied to any microarray dataset and any target gene. We applied this method to microarray data generated from wine fermentations and selected NSF1, which encodes a C2H2 zinc finger-type transcription factor, as the target gene. The validity of the method was verified by accurate identifications of the previously known functional roles of NSF1. In addition, we identified and verified potential new functions for this gene; specifically, NSF1 is a negative regulator for the expression of sulfur metabolism genes, the nuclear localization of Nsf1 protein (Nsf1p) is controlled in a sulfur-dependent manner, and the transcription of NSF1 is regulated by Met4p, an important transcriptional activator of sulfur metabolism genes. The inter-disciplinary approach adopted here highlighted the accuracy and relevancy of the ICC method in mining for novel gene functions using complex microarray datasets with a limited number of samples.

The fermentation stress response protein Aaf1p/Yml081Wp regulates acetate production in Saccharomyces cerevisiae.

The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of ALD4 and ALD6, which encode a cytosolic acetaldehyde dehydrogenase (ACDH) and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of ALD6, YML081W had no effect on acetic acid levels, suggesting that this transcription factor's effects are mediated primarily through this gene. lacZ reporter assays revealed that Yml081wp stimulates ALD6 transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in Saccharomyces cerevisiae. We propose AAF1 as a gene name for the YML081W ORF.

A functional peroxisome proliferator-activated receptor-gamma ligand-binding domain is not required for adipogenesis.

The nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) is the central regulator of adipogenesis. Although it is the target for several drugs that function as agonist activators, a high affinity endogenous ligand for this receptor that is involved in regulating adipogenesis has yet to be identified. Here, we investigated the requirement for ligand activation of PPARgamma in fat cell differentiation, taking advantage of a natural mutant of this receptor that does not bind or become activated by any known natural or synthetic ligand. When ectopically expressed in PPARgamma-null fibroblasts, this Q286P allele was able to strongly promote morphological adipogenesis, without any significant difference compared with wild-type PPARgamma. In addition, no significant differences were found in the expression of several adipogenic genes between the wild-type and Q286P mutant alleles. To extend our studies to an in vivo setting, we performed subcutaneous injections of PPARgamma-expressing fibroblasts into nude mice. We found that both wild-type and Q286P mutant-expressing fibroblasts were able to generate fat pads in the mice. These results suggest that the binding and activation of PPARgamma by agonist ligands may not be required for adipogenesis under physiological conditions.