Systemic administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque CNS.

BACKGROUND: Adeno-associated virus (AAV) vectors are a promising vehicle for noninvasive gene delivery to the central nervous system via intravenous infusion. However, naturally occurring serotypes have a limited ability to transduce the brain, and translating engineered capsids from mice to nonhuman primates has proved challenging. METHODS: In this study, we use an mRNA-based directed-evolution strategy in multiple strains of mice as well as a de novo selection in cynomolgus macaques to identify families of engineered vectors with increased potency in the brain and decreased tropism for the liver. FINDINGS: We compare the transgene expression capabilities of several engineered vectors and show that while some of our novel macaque-derived variants significantly outperform AAV9 in transducing the macaque brain following systemic administration, mouse-derived variants-both those identified in this study and those reported by other groups-universally do not. CONCLUSIONS: Together, the results of this work introduce a class of primate-derived engineered AAV capsids with increased therapeutic potential and highlight the critical need for using appropriate animal models to both identify and evaluate novel AAVs intended for delivery to the human central nervous system. FUNDING: This work was funded primarily through an anonymous philanthropic gift to the P.C.S. lab at the Broad Institute of MIT and Harvard and by a grant from the Howard Hughes Medical Institute to P.C.S.

Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.

Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.

The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer.

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.

Mapping choline metabolites in normal and transformed cells.

INTRODUCTION: Choline is an essential human nutrient that is particular important for proliferating cells, and altered choline metabolism has been associated with cancer transformation. Yet, the various metabolic fates of choline in proliferating cells have not been investigated systematically. OBJECTIVES: This study aims to map the metabolic products of choline in normal and cancerous proliferating cells. METHODS: We performed 13C-choline tracing followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of metabolic products in normal and in vitro-transformed (tumor-forming) epithelial cells, and also in tumor-derived cancer cell lines. Selected metabolites were quantified by internal standards. RESULTS: Untargeted analysis revealed 121 LCMS peaks that were 13C-labeled from choline, including various phospholipid species, but also previously unknown products such as monomethyl- and dimethyl-ethanolamines. Interestingly, we observed formation of betaine from choline specifically in tumor-derived cells. Expression of choline dehydrogenase (CHDH), which catalyzes the first step of betaine synthesis, correlated with betaine synthesis across the cell lines studied. RNAi silencing of CHDH did not affect cell proliferation, although we observed an increased fraction of G2M phase cells with some RNAi sequences, suggesting that CHDH and its product betaine may play a role in cell cycle progression. Betaine cell concentration was around 10 µM, arguing against an osmotic function, and was not used as a methyl donor. The function of betaine in these tumor-derived cells is presently unknown. CONCLUSION: This study identifies novel metabolites of choline in cancer and normal cell lines, and reveals altered choline metabolism in cancer cells.

Mapping metabolic oscillations during cell cycle progression.

Proliferating cells must synthesize a wide variety of macromolecules while progressing through the cell cycle, but the coordination between cell cycle progression and cellular metabolism is still poorly understood. To identify metabolic processes that oscillate over the cell cycle, we performed comprehensive, non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) based metabolomics of HeLa cells isolated in the G1 and SG2M cell cycle phases, capturing thousands of diverse metabolite ions. When accounting for increased total metabolite abundance due to cell growth throughout the cell cycle, 18% of the observed LC-HRMS peaks were at least twofold different between the stages, consistent with broad metabolic remodeling throughout the cell cycle. While most amino acids, phospholipids, and total ribonucleotides were constant across cell cycle phases, consistent with the view that total macromolecule synthesis does not vary across the cell cycle, certain metabolites were oscillating. For example, ribonucleotides were highly phosphorylated in SG2M, indicating an increase in energy charge, and several phosphatidylinositols were more abundant in G1, possibly indicating altered membrane lipid signaling. Within carbohydrate metabolism, pentose phosphates and methylglyoxal metabolites were associated with the cycle. Interestingly, hundreds of yet uncharacterized metabolites similarly oscillated between cell cycle phases, suggesting previously unknown metabolic activities that may be synchronized with cell cycle progression, providing an important resource for future studies.

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG2M Phase.

Alterations in cell-cycle regulation and cellular metabolism are associated with cancer transformation, and enzymes active in the committed cell-cycle phase may represent vulnerabilities of cancer cells. Here, we map metabolic events in the G1 and SG2M phases by combining cell sorting with mass spectrometry-based isotope tracing, revealing hundreds of cell-cycle-associated metabolites. In particular, arginine uptake and ornithine synthesis are active during SG2M in transformed but not in normal cells, with the mitochondrial arginase 2 (ARG2) enzyme as a potential mechanism. While cancer cells exclusively use ARG2, normal epithelial cells synthesize ornithine via ornithine aminotransferase (OAT). Knockdown of ARG2 markedly reduces cancer cell growth and causes G2M arrest, while not inducing compensation via OAT. In human tumors, ARG2 is highly expressed in specific tumor types, including basal-like breast tumors. This study sheds light on the interplay between metabolism and cell cycle and identifies ARG2 as a potential metabolic target.

Choline metabolite, trimethylamine N-oxide (TMAO), is associated with inflammation in psoriatic arthritis.

OBJECTIVES: Dietary intake of choline has been linked to systemic inflammation through the microbial production of two metabolites, trimethylamine (TMA) and trimethylamine-N-oxide (TMAO). Herein we explore the association between choline metabolites and inflammation in psoriatic arthritis (PsA) patients. METHODS: Thirty-eight patients with PsA, all of whom satisfied the CASPAR classification criteria for PsA, were studied. Outcomes reflecting the activity of peripheral arthritis as well as skin psoriasis, Disease Activity Score (DAS)28, Clinical Disease Index (CDAI) and Body Surface Area (BSA) were assessed. Serum concentration of choline metabolites (choline, TMA, TMAO, betaine and carnitine) were determined by LC-MS, and metabolite levels associated with disease scores. RESULTS: Among the 38 PsA patients included, the mean DAS28PCR was 2.74±1.29. Twenty-seven patients had active skin disease, with an average BSA of 7.2±16.22. TMAO, but not TMA or choline, significantly correlated with measures of disease activity for both skin and peripheral joints. CONCLUSIONS: In our cohort, only TMAO, but not TMA, choline, betaine or carnitine, was associated with inflammation in PsA patients, establishing a mechanistic link between TMAO and PsA phenotypes. Future studies will explore the modulation of TMAO and disease severity in PsA.

Gabapentin Can Suppress Cell Proliferation Independent of the Cytosolic Branched-Chain Amino Acid Transferase 1 (BCAT1).

The metabolism of branched-chain amino acids (BCAA) has recently been implicated in the growth of several cancer cell types. Gabapentin, a synthetic amino acid, is commonly used in high concentrations in this context to inhibit the cytosolic branched-chain amino acid transferase (BCAT1) enzyme. Here, we report that 10 mM gabapentin reduces the growth of HCT116 cells, which have an active branched-chain amino acid transferase but express very low levels of BCAT1, and presumably rely on the mitochondrial BCAT2 enzyme. Gabapentin did not affect transamination of BCAA to branched-chain keto acids (BCKA) in HCT116 cells, nor the reverse formation of BCAA from BCKA, indicating that the branched-chain amino acid transaminase is not inhibited. Moreover, the growth-inhibitory effect of gabapentin could not be rescued by supplementation with BCKA, and this was not due to the lack of uptake of BCKA, indicating that other effects of gabapentin are important. An untargeted LC-MS analysis of gabapentin-treated cells revealed a marked depletion of branched-chain carnitines. These results demonstrate that gabapentin at high concentrations can inhibit cell proliferation without affecting BCAT1 and may affect mitochondrial BCKA catabolism.

Profiling the Metabolism of Human Cells by Deep 13C Labeling.

Studying metabolic activities in living cells is crucial for understanding human metabolism, but facile methods for profiling metabolic activities in an unbiased, hypothesis-free manner are still lacking. To address this need, we here introduce the deep-labeling method, which combines a custom 13C medium with high-resolution mass spectrometry. A proof-of-principle study on human cancer cells demonstrates that deep labeling can identify hundreds of endogenous metabolites as well as active and inactive pathways. For example, protein and nucleic acids were almost exclusively de novo synthesized, while lipids were partly derived from serum; synthesis of cysteine, carnitine, and creatine was absent, suggesting metabolic dependencies; and branched-chain keto acids (BCKAs) were formed and metabolized to short-chain acylcarnitines, but did not enter the tricarboxylic acid cycle. Remarkably, BCKAs could substitute for essential amino acids to support growth. The deep-labeling method may prove useful to map metabolic phenotypes across a range of cell types and conditions.

Male lifespan extension with 17-α estradiol is linked to a sex-specific metabolomic response modulated by gonadal hormones in mice.

Longevity in mammals is influenced by sex, and lifespan extension in response to anti-aging interventions is often sex-specific, although the mechanisms underlying these sexual dimorphisms are largely unknown. Treatment of mice with 17-α estradiol (17aE2) results in sex-specific lifespan extension, with an increase in median survival in males of 19% and no survival effect in females. Given the links between lifespan extension and metabolism, we performed untargeted metabolomics analysis of liver, skeletal muscle and plasma from male and female mice treated with 17aE2 for eight months. We find that 17aE2 generates distinct sex-specific changes in the metabolomic profile of liver and plasma. In males, 17aE2 treatment raised the abundance of several amino acids in the liver, and this was further associated with elevations in metabolites involved in urea cycling, suggesting altered amino acid metabolism. In females, amino acids and urea cycling metabolites were unaffected by 17aE2. 17aE2 also results in male-specific elevations in a second estrogenic steroid-estriol-3-sulfate-suggesting different metabolism of this drug in males and females. To understand the underlying endocrine causes for these sexual dimorphisms, we castrated males and ovariectomized females prior to 17aE2 treatment, and found that virtually all the male-specific metabolite responses to 17aE2 are inhibited or reduced by male castration. These results suggest novel metabolic pathways linked to male-specific lifespan extension and show that the male-specific metabolomic response to 17aE2 depends on the production of testicular hormones in adult life.