In vivo genome editing at the albumin locus to treat methylmalonic acidemia

Abstract

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.

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

“…Because SIRT5 is mitochondrially localized and has a preference for negatively charged acylation modifications including malonylation ( 27 ), we posited that a loss of SIRT5 activity would likely result in increased disease severity in MMA due to an escalation of methylmalonylation. Using a genetic approach, we generated a double mutant line mouse line, Sirt5 −/− ; Mmut G715V/G715V , by breeding mice with a partial deficiency form of isolated MMA ( Mmut G715V/G715V ) ( 68 ) with those carrying an Sirt5 loss of function allele ( 69 ).…”

“…For the Mmut −/− ;Tg INS-MCK-Mmut and Mmut −/− ;Tg INS-Alb-Mmut mouse lines, see the methods as previously described in ( 32 , 58 ). For the Sirt5 −/− ;Mmut G715V/G715V mouse line, B6;129- Sirt5 tm1Fwa/J mice from the Jackson Laboratory (JAX, stock #012757) ( 69 ) were crossed to the Mmut G715V/G715V ( 68 ) line to generate mice heterozygous for both mutant alleles. Heterozygous mice were then crossed to generate Sirt5 +/− ; Mmut +/G715V and Sirt5 −/− ; Mmut +/G715V females and Sirt5 −/− ; Mmut G715V/G715V males, which were then crossed to the Sirt5 −/− ; Mmut +/G715V females to generate double-mutant Sirt5 −/− ; Mmut G715V/G715V mice.…”

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia

“…Control of the metabolic crisis requires expansion of corrected hepatocytes to populate a significant fraction of the liver. Interestingly, a recent report shows that using exogenous nucleases to increase the initial editing frequency can significantly reduce the treatment-to-efficacy time window [ 29 ].…”

Treatment of metabolic disorders using genomic technologies: Lessons from methylmalonic acidemia

“…In the mouse Albumin gene, we identified and tested a Cas9 guide targeting the start codon of Alb, as well as another in the first intron, and designed corresponding donor AAVs that contains Alb homology arms flanking a codon-optimized human MMUT cDNA (Figure 2D-F). 50 We then tested various editing approaches in newly constructed, transgene-free knockin MMA mouse models that recapitulate severe (mut o ÀMmut p.R106C/p.R106C mice) and partial (mut À ÀMmut p.G715V/p.G715V ) deficiency forms of the disorder (Table 1), and compared the results to conventional AAV gene addition therapy with either AAV8 or AAV9 vectors. 50 While all editing strategies were successful, the 5 0 ATG targeted approach merits further discussion (Figure 2D).…”

“…50 We then tested various editing approaches in newly constructed, transgene-free knockin MMA mouse models that recapitulate severe (mut o ÀMmut p.R106C/p.R106C mice) and partial (mut À ÀMmut p.G715V/p.G715V ) deficiency forms of the disorder (Table 1), and compared the results to conventional AAV gene addition therapy with either AAV8 or AAV9 vectors. 50 While all editing strategies were successful, the 5 0 ATG targeted approach merits further discussion (Figure 2D). Mmut p.R106C/p.R106C mice treated with either Cas9 and a guide targeting the ATG of Alb and the donor AAV, or with only the donor AAV, exhibited improved survival compared to untreated mutant mice.…”