We used exome sequencing to identify the genetic basis of combined malonic and methylmalonic aciduria (CMAMMA). We sequenced the exome of an individual with CMAMMA and followed up with sequencing of eight additional affected individuals (cases). This included one individual who was identified and diagnosed by searching an exome database. We identify mutations in ACSF3, encoding a putative methylmalonyl-CoA and malonyl-CoA synthetase as a cause of CMAMMA. We also examined a canine model of CMAMMA, which showed pathogenic mutations in a predicted ACSF3 ortholog. ACSF3 mutant alleles occur with a minor allele frequency of 0.0058 in ∼1,000 control individuals, predicting a CMAMMA population incidence of ∼1:30,000. ACSF3 deficiency is the first human disorder identified as caused by mutations in a gene encoding a member of the acyl-CoA synthetase family, a diverse group of evolutionarily conserved proteins, and may emerge as one of the more common human metabolic disorders.

Methylmalonic acidemia (MMA), a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition.

Adeno-associated viral (AAV) gene therapy has shown great promise as an alternative treatment for metabolic disorders managed using liver transplantation, but remains limited by transgene loss and genotoxicity. Our study aims to test an AAV vector with a promoterless integrating cassette, designed to provide sustained hepatic transgene expression and reduced toxicity in comparison to canonical AAV therapy.

Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating metabolites, transgene expression, and whole animal propionate oxidation were examined as outcome parameters after gene therapy. One-hundred percent of the untreated Mut(-/-) mice (n = 58) died by day of life (DOL) 72, whereas >95% of the adeno-associated virus-treated Mut(-/-) mice (n = 27) have survived for > or = 1 year. Despite a gradual loss of transgene expression and elevated circulating metabolites in the treated Mut(-/-) mice, the animals are indistinguishable from unaffected control littermates in size and activity levels. These experiments provide the first definitive evidence that gene therapy will have clinical utility in the treatment of MMA and support the development of gene therapy for other organic acidemias.

Adeno-associated viral (AAV) vectors have emerged as the preferred platform for in vivo gene transfer because of their combined efficacy and safety. However, insertional mutagenesis with the subsequent development of hepatocellular carcinomas (HCCs) has been recurrently noted in newborn mice treated with high doses of AAV, and more recently, the association of wild-type AAV integrations in a subset of human HCCs has been documented. Here, we address, in a comprehensive, prospective study, the long-term risk of tumorigenicity in young adult mice following delivery of single-stranded AAVs targeting liver. HCC incidence in mice treated with therapeutic and reporter AAVs was low, in contrast to what has been previously documented in mice treated as newborns with higher doses of AAV. Specifically, HCCs developed in 6 out 76 of AAV-treated mice, and a pathogenic integration of AAV was found in only one tumor. Also, no evidence of liver tumorigenesis was found in juvenile AAV-treated mucopolysaccharidosis type VI (MPS VI) cats followed as long as 8 years after vector administration. Together, our results support the low risk of tumorigenesis associated with AAV-mediated gene transfer targeting juvenile/young adult livers, although constant monitoring of subjects enrolled in AAV clinical trial is advisable.

Methylmalonic acidemia (MMA) is a severe metabolic disorder most commonly caused by a mutation in the methylmalonyl-CoA mutase (MMUT) gene. Patients with MMA experience multisystemic disease manifestations and remain at risk for neurological disease progression, even after liver transplantation. Therefore, delivery of MMUT to the central nervous system (CNS) may provide patients with neuroprotection and, perhaps, therapeutic benefits. To specifically target the brain, we developed a neurotropic PHP.eB vector that used a CaMKII neuro-specific promoter to restrict the expression of the MMUT transgene in the neuraxis and delivered the adeno-associated virus (AAV) to mice with MMA. The PHP.eB vector transduced cells in multiple brain regions, including the striatum, and enabled high levels of expression of MMUT in the basal ganglia. Following the CNS-specific correction of MMUT expression, disease-related metabolites methylmalonic acid and 2-methylcitrate were significantly (p < 0.02) decreased in serum of treated MMA mice. Our results show that targeting MMUT expression to the CNS using a neurotropic capsid can decrease the circulating metabolite load in MMA and further highlight the benefit of extrahepatic correction for disorders of organic acid metabolism.

Crohn's disease (CD) is a major subtype of inflammatory bowel disease (IBD), a spectrum of chronic intestinal disorders caused by dysregulated immune responses to gut microbiota. Although transcriptional and functional changes in a number of immune cell types have been implicated in the pathogenesis of IBD, the cellular interactions and signals that drive these changes have been less well-studied.

Tissue-resident memory CD8+ T (TRM) cells are a subset of memory T cells that play a critical role in limiting early pathogen spread and controlling infection. TRM cells exhibit differences across tissues, but their potential heterogeneity among distinct anatomic compartments within the small intestine and colon has not been well recognized. Here, by analyzing TRM cells from the lamina propria and epithelial compartments of the small intestine and colon, we showed that intestinal TRM cells exhibited distinctive patterns of cytokine and granzyme expression along with substantial transcriptional, epigenetic, and functional heterogeneity. The T-box transcription factor Eomes, which represses TRM cell formation in some tissues, exhibited unexpected context-specific regulatory roles in supporting the maintenance of established TRM cells in the small intestine, but not in the colon. Taken together, these data provide previously unappreciated insights into the heterogeneity and differential requirements for the formation vs. maintenance of intestinal TRM cells.

Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of ∼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9-Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation.

We have utilized Caenorhabditis elegans to study human methylmalonic acidemia. Using bioinformatics, a full complement of mammalian homologues for the conversion of propionyl-CoA to succinyl-CoA in the genome of C. elegans, including propionyl-CoA carboxylase subunits A and B (pcca-1, pccb-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), co(I)balamin adenosyltransferase (mmab-1), MMACHC (cblc-1), methylmalonyl-CoA epimerase (mce-1) and methylmalonyl-CoA mutase (mmcm-1) were identified. To verify predictions that the entire intracellular adenosylcobalamin metabolic pathway existed and was functional, the kinetic properties of the C. elegans mmcm-1 were examined. RNA interference against mmcm-1, mmab-1, mmaa-1 in the presence of propionic acid revealed a chemical phenotype of increased methylmalonic acid; deletion mutants of mmcm-1, mmab-1 and mce-1 displayed reduced 1-[(14)C]-propionate incorporation into macromolecules. The mutants produced increased amounts of methylmalonic acid in the culture medium, proving that a functional block in the pathway caused metabolite accumulation. Lentiviral delivery of the C. elegans mmcm-1 into fibroblasts derived from a patient with mut(o) class methylmalonic acidemia could partially restore propionate flux. The C. elegans mce-1 deletion mutant demonstrates for the first time that a lesion at the epimerase step of methylmalonyl-CoA metabolism can functionally impair flux through the methylmalonyl-CoA mutase pathway and suggests that malfunction of MCEE may cause methylmalonic acidemia in humans. The C. elegans system we describe represents the first lower metazoan model organism of mammalian propionate spectrum disorders and demonstrates that mass spectrometry can be employed to study a small molecule chemical phenotype in C. elegans RNAi and deletion mutants.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials.

Niemann-Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1 m1N/m1N mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1 m1N/m1N mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1 m1N/m1N mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.

The use of adeno-associated virus (AAV) as a gene therapy vector has been approved recently for clinical use and has demonstrated efficacy in a growing number of clinical trials. However, the safety of AAV as a vector has been challenged by a single study that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. Most studies have not noted genotoxicity following AAV-mediated gene delivery; therefore, the possibility that there is an association between AAV and HCC is controversial. Here, we performed a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the RNA imprinted and accumulated in nucleus (Rian) locus, and the resulting overexpression of proximal microRNAs and retrotransposon-like 1 (Rtl1) were associated with HCC. In addition, we demonstrated that the AAV vector dose, enhancer/promoter selection, and the timing of gene delivery are all critical factors for determining HCC incidence after AAV gene delivery. Together, our results define aspects of AAV-mediated gene therapy that influence genotoxicity and suggest that these features should be considered for design of both safer AAV vectors and gene therapy studies.

The activating receptor NKG2D and its ligands are recognized as a potent immune axis that controls tumor growth and microbial infections. With regards to cancer surveillance, various studies have demonstrated the antitumor function mediated by NKG2D on natural killer cells and on conventional and unconventional T cells. The use of NKG2D-deficient mice established the importance of NKG2D in delaying tumor development in transgenic mouse models of cancer. However, we recently demonstrated an unexpected, flip side to this coin, the ability for NKG2D to contribute to tumor growth in a model of inflammation-driven liver cancer. With a focus on the liver, here, we review current knowledge of NKG2D-mediated tumor surveillance and discuss evidence supporting a dual role for NKG2D in cancer immunity. We postulate that in certain advanced cancers, expression of ligands for NKG2D can drive cancer progression rather than rejection. We propose that the nature of the microenvironment within and surrounding tumors impacts the outcome of NKG2D activation. In a form of autoimmune attack, NKG2D promotes tissue damage, mostly in the inflamed tissue adjacent to the tumor, facilitating tumor progression while being ineffective at rejecting transformed cells in the tumor bed.

Methylmalonic acidemia (MMA) is a metabolic disorder most commonly caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Although adeno-associated viral (AAV) gene therapy has been effective at correcting the disease phenotype in MMA mouse models, clinical translation may be impaired by loss of episomal transgene expression and magnified by the need to treat patients early in life. To achieve permanent correction, we developed a dual AAV strategy to express a codon-optimized MMUT transgene from Alb and tested various CRISPR-Cas9 genome-editing vectors in newly developed knockin mouse models of MMA. For one target site in intron 1 of Alb, we designed rescue cassettes expressing MMUT behind a 2A-peptide or an internal ribosomal entry site sequence. A second guide RNA targeted the initiator codon, and the donor cassette encompassed the proximal albumin promoter in the 5' homology arm. Although all editing approaches were therapeutic, targeting the start codon of albumin allowed the use of a donor cassette that also functioned as an episome and after homologous recombination, even without the expression of Cas9, as an integrant. Targeting the albumin locus using these strategies would be effective for other metabolic disorders where early treatment and permanent long-term correction are needed.

Hyperbaric oxygen therapy (HBOT) is a promising treatment for moderate-to-severe ulcerative colitis. However, our current understanding of the host and microbial response to HBOT remains unclear. This study examined the molecular mechanisms underpinning HBOT using a multi-omic strategy.

During an immune response to microbial infection, CD8+ T cells give rise to short-lived effector cells and memory cells that provide sustained protection. Although the transcriptional programs regulating CD8+ T cell differentiation have been extensively characterized, the role of long noncoding RNAs (lncRNAs) in this process remains poorly understood. Using a functional genetic knockdown screen, we identified the lncRNA Malat1 as a regulator of terminal effector cells and the terminal effector memory (t-TEM) circulating memory subset. Evaluation of chromatin-enriched lncRNAs revealed that Malat1 grouped with trans lncRNAs that exhibit increased RNA interactions at gene promoters and gene bodies. Moreover, we observed that Malat1 was associated with increased H3K27me3 deposition at a number of memory cell-associated genes through a direct interaction with Ezh2, thereby promoting terminal effector and t-TEM cell differentiation. Our findings suggest an important functional role of Malat1 in regulating CD8+ T cell differentiation and broaden the knowledge base of lncRNAs in CD8+ T cell biology.