Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function.

Aberrant methylmalonylation underlies methylmalonic acidemia and is attenuated by an engineered sirtuin.

Organic acidemias such as methylmalonic acidemia (MMA) are a group of inborn errors of metabolism that typically arise from defects in the catabolism of amino and fatty acids. Accretion of acyl-CoA species is postulated to underlie disease pathophysiology, but the mechanism(s) remain unknown. Here, we surveyed hepatic explants from patients with MMA and unaffected donors, in parallel with samples from various mouse models of methylmalonyl-CoA mutase deficiency.

genome editing at the albumin locus to treat methylmalonic acidemia.

Methylmalonic acidemia (MMA) is a metabolic disorder most commonly caused by mutations in the methylmalonyl-CoA mutase () 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 transgene from and tested various CRISPR-Cas9 genome-editing vectors in newly developed knockin mouse models of MMA.

Promoterless, Nuclease-Free Genome Editing Confers a Growth Advantage for Corrected Hepatocytes in Mice With Methylmalonic Acidemia.

Background And Aims: 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.

Approach And Results: Our AAV vector was designed to insert a methylmalonyl-CoA mutase (MMUT) transgene into the 3' end of the albumin locus and tested in mouse models of methylmalonic acidemia (MMA).

Systemic Messenger RNA Therapy as a Treatment for Methylmalonic Acidemia.

Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs).

Genome editing for inborn errors of metabolism: advancing towards the clinic.

Inborn errors of metabolism (IEM) include many disorders for which current treatments aim to ameliorate disease manifestations, but are not curative. Advances in the field of genome editing have recently resulted in the in vivo correction of murine models of IEM. Site-specific endonucleases, such as zinc-finger nucleases and the CRISPR/Cas9 system, in combination with delivery vectors engineered to target disease tissue, have enabled correction of mutations in disease models of hemophilia B, hereditary tyrosinemia type I, ornithine transcarbamylase deficiency, and lysosomal storage disorders.

Three-dimensional macroporous graphene scaffolds for tissue engineering.

The assembly of carbon nanomaterials into three-dimensional (3D) porous scaffolds is critical to harness their unique physiochemical properties for tissue engineering and regenerative medicine applications. In this study, we report the fabrication, characterization, and in vitro cytocompatibility of true 3D (>1 mm in all three dimensions), macroscopic (3-8 mm in height and 4-6 mm in diameter), chemically cross-linked graphene scaffolds prepared via radical initiated thermal cross-linking of single- and multiwalled graphene oxide nanoribbons (SWGONRs and MWGONRs). SWGONR and MWGONR scaffolds possess tunable porosity (∼65-80%) and interconnected macro-, micro-, and nanoscale pores.