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Published in final edited form as: Mol Genet Metab. 2022 Jul 4;137(1-2):1–8. doi: 10.1016/j.ymgme.2022.06.011

Growth advantage of corrected hepatocytes in a juvenile model of methylmalonic acidemia following liver directed adeno-associated viral mediated nuclease-free genome editing

Leah E Venturoni a,1, Randy J Chandler a,1, Jing Liao b, Victoria Hoffmann c, Nikhil Ramesh b, Susana Gordo b, Nelson Chau b, Charles P Venditti a,*
PMCID: PMC9872049  NIHMSID: NIHMS1826136  PMID: 35868241

Abstract

Methylmalonic acidemia (MMA) is a rare and severe inherited metabolic disease typically caused by mutations of the methylmalonyl-CoA mutase (MMUT) gene. Despite medical management, patients with MMA experience frequent episodes of metabolic instability, severe morbidity, and early mortality. In several preclinical studies, systemic gene therapy has demonstrated impressive improvement in biochemical and clinical phenotypes of MMA murine models. One approach uses a promoterless adeno-associated viral (AAV) vector that relies upon homologous recombination to achieve site-specific in vivo gene addition of MMUT into the last coding exon of albumin (Alb), generating a fused Alb-MMUT transcript after successful editing. We have previously demonstrated that nuclease-free AAV mediated Alb editing could effectively treat MMA mice in the neonatal period and noted that hepatocytes had a growth advantage after correction. Here, we use a transgenic knock-out mouse model of MMA that recapitulates severe clinical and biochemical symptoms to assess the benefits of Alb editing in juvenile animals. As was first noted in the neonatal gene therapy studies, we observe that gene edited hepatocytes in the MMA mice treated as juveniles exhibit a growth advantage, which allows them to repopulate the liver slowly but dramatically by 8–10 months post treatment, and subsequently manifest a biochemical and enzymatic response. In conclusion, our results suggest that the benefit of AAV mediated nuclease-free gene editing of the Alb locus to treat MMA could potentially be therapeutic for older patients.

Keywords: genome editing, gene therapy, methylmalonic acidemia, MMA, fibroblast growth factor 21, FGF21, organic acidemia, adeno-associated virus, AAV, gene editing, selective growth advantage, albumin

1. INTRODUCTION

Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism most commonly caused by a deficiency of methylmalonyl-CoA mutase (MMUT) [1]. MMUT is a mitochondrially localized enzyme responsible for converting methylmalonyl-CoA to succinyl-CoA. A reduced or complete loss of enzymatic activity results in elevated methylmalonic acid levels in all tissues. Clinically, MMA is a heterogenous multiorgan disease, and the clinical course is characterized by recurrent episodes of life-threatening metabolic crises [2]. Medical management is focused on the dietary restriction of protein and symptomatic management [3–6]. The failure of conventional therapy to improve the outcomes seen in severely affected patients has led to the adoption of elective liver transplantation as a surgical treatment for MMA, with some centers successfully using living related donors to provide the liver to patients less than 1 year of age [7–9]. Although liver transplantation exposes patients to procedural risks and requires life-long immunosuppression, it can prevent metabolic instability and improve quality of life and illustrates the benefits of restored hepatic MMUT activity [9–14]. The promising clinical benefits seen with liver transplantation in MMA patients have helped inspire the development of gene-based therapies to treat MMA in animal models, including AAV based approaches.

Previous work has demonstrated that conventional AAV mediated gene delivery can effectively rescue neonatal mice with the most severe form of MMA [15–21]. However, a rapid loss of hepatic transgene expression was observed, and most of the treated animals developed hepatocellular carcinoma from AAV-mediated insertional mutagenesis of the Rian locus [22, 23]. For these reasons, we previously developed a promoterless, nuclease free AAV vector (AAVDJ-Alb-2A-MMUT) designed to express MMUT after editing into Alb [24–28]. The therapeutic benefit of this approach relies upon successful homologous recombination to target the MMUT transgene into the native mouse safe harbor Alb locus, just upstream of the stop codon at the 3’ end of this gene (Fig. 1A). After on-target integration, a fused Alb-2A-MMUT mRNA is produced under the regulation of the native Alb promoter. The 2A peptide sequence mediates ribosomal skipping [29–32] and allows the Alb-2A-MMUT mRNA to produce two distinct proteins: ALB-2A and MMUT. Edited Alb alleles will produce a circulating Alb-2A protein which can serve as a biomarker of successful recombination.

Fig 1. AAVDJ-Alb-2A-MMUT treatment and response in MMA mice.

Fig 1.

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A. A schematic of AAVDJ-Alb-2A-MMUT integration and expression. An AAV containing the 2A-MMUT sequenced flanked by arms of homology to either side of the stop codon of Alb is delivered to hepatocytes in the liver-tropic AAVDJ capsid. Homologous recombination between the native murine Alb locus and the AAV generates a fused Alb-2A-MMUT allele under the control of the native Alb promoter, producing a fused mRNA. 2A mediated self-cleavage produces two distinct proteins, ALB-2A and MMUT, where the start methionine of MMUT has been replaced with proline. Only edited alleles will produce ALB-2A. B. Survival of mice treated at two months of age (n=11) was not significantly different from untreated MMA mice (n=24) (p=0.57; (MantelCox) test). C. Treated MMA mice (n= 11, 10, 7, 4, 4, 3, 2, 3, 3) have reduced levels of plasma methylmalonic acid (pMMA) compared to untreated MMA mice (n= 11, 10, 5, 8, 5, 0, 7, 0, 7) with a significant reduction at 4 and 10 months of age (*p<0.05; unpaired t-test). The dotted line at 2 months represents when the mice were treated. Error bars are ± the SEM. D. Treated MMA mice (n=8) had significantly reduced plasma FGF21 levels compared to untreated MMA mice (n=5) (*p<0.0001; unpaired t-test).

We previously reported the efficacy, durability, and safety of AAVDJ-Alb-2A-MMUT in two severe mouse models of MMA treated systemically as neonates [28]. Neonatal treated MMA mice had durable MMUT expression, improved growth with no genotoxicity, in addition to decrease plasma levels of disease biomarkers, FGF21, and methylmalonic acid. Less than 1 percent of the hepatocytes were edited at 2–3 months post-treatment in the MMA mice, which was expected for a gene editing strategy that relies on homologous recombination. However, a significantly higher percentage (12–20%) of corrected hepatocytes was observed at 11–15 months post treatment in the MMA mice but a similar increase was not observed in identically treated control littermates, suggesting the corrected hepatocytes have a selective growth advantage in the setting of MMA. Hepatic MMUT protein expression and the percentage of MMUT mRNA expressing hepatocytes were both significantly increased at 11–15 months post treatment, which further supported the observation of a selective growth advantage conferred upon diseased hepatocytes after successful gene editing. The correction of the underlying disease related mitochondrial dysfunction that has been described in both the hepatocytes of MMA mice and patients is hypothesized to be responsible for this growth advantage.

Two important questions that our previous studies did not address are whether this novel gene editing platform to treat MMA could be an effective treatment in older mice and if selection and expansion of corrected MMA hepatocytes might be observed. Here, we demonstrate that AAV-mediated nuclease-free genome editing into albumin in a juvenile hypomorphic mouse model of MMA produces a durable and therapeutic expression of MMUT without genotoxicity. In addition, we show that the selective growth advantage previously observed in MMA mice treated as neonates also occurs in older treated MMA animals.

2. MATERIALS AND METHODS:

2.1. Animal Studies.

Animal work was performed in accordance with the National Institutes of Health guide for the care and use of laboratory animals. The mouse model of MMA, Mmut–/–;TgINS-MCK-Mmut, is maintained on a mixed C57BL/6 × 129SV/Ev × FvBN genetic background as previously described [21]. Mice were fed standard mouse chow (Prolab® RMH 1800) and maintained without special dietary or environmental considerations. Treated mice received a single AAV dose of 1e12 vector genomes (VG) per mouse (~7e13 VG/kg) via retroorbital injection, injected between 44 and 65 days of life. Mice were bled via retro-orbital sinus plexus sampling monthly and frequently weighed. Treated mice were predominantly female (n=10 of 11 treated animals).

2.2. Plasmid Vector and rAAV production.

AAV-Alb-2A-MMUT was generated by replacing the FIX cDNA with a human codon-optimized MMUT cDNA from the pAB288 plasmid [24] as previously described [28]. AAVDJ vectors were produced by triple transfection with Ca3(PO4)2 followed by CsCl gradient purification [33] and titered as previously described [34].

2.3. Methylmalonic Acid Measurement.

Methylmalonic acid was measured in plasma using gas chromatography-mass spectrometry with stable isotopic internal calibration, as previously described [35].

2.4. Western Blot Analysis.

Samples were prepared and run according to standard protocols. The following antibodies were used for the detection of MMUT (Abcam, ab134956) and ß-Actin (Proteintech, 66009–1-Ig) in conjunction with the following secondary antibodies; (LI-COR, 925–32211), and (LI-COR, 926–68072). Quantification of western blot bands was performed using Image Studio Lite Version 5.2.

2.5. RNA in situ hybridization.

RNAscope probes to detect the human codon-optimized MMUT RNA were designed by ACDBio using their proprietary technology and do not cross-react with other mRNAs in the murine transcriptome. Livers were fixed in 4% PFA or 10% NBF and processed into paraffin blocks. Five-micron sections were cut and stained with RNAScope 2.5 HD Assay-Brown (ACDBio 322300) following the manufacturer’s instructions. Slide images were captured using an Aperio scanner and quantification was performed using QuPath [36]. Cell detection was performed by detecting the nucleus based on optical density sum. For probe quantification, hepatocytes were divided into the following categories according to probe detection within the cytoplasm: negative; 1+, 1–9 puncta; 2+, 10–19 puncta; or 3+, >20 puncta. Nuclear staining was excluded. An H-score was calculated as follows [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)] to generate a score for each sample between 0 and 300 [37].

2.6. Histology and Hepatocellular Carcinoma Screening.

Livers were visually inspected for abnormalities at necropsy, then fixed in formalin, embedded, serially sectioned, stained with hematoxylin and eosin, and reviewed by a veterinary pathologist.

2.7. Vector Genome Quantitation.

Genomic DNA from liver samples was extracted using DNeasy Blood & Tissue Kit (Qiagen; 69506). ddPCR was performed according to the manufacturer’s recommendations for the BioRad QX200 AutoDG ddPCR system using 10 ng or 20 ng of DNA as input and the following probes: BIO-RAD ddPCR CNV assay Gapdh (Cat # 10042961, dMumCNS300520369) and MMUT (Cat # 10042958 dCNS513322846).

2.8. Albumin Integration Assay.

Genomic DNA from liver samples was extracted using DNeasy Blood & Tissue Kit (Qiagen; 69504). Genomic integration was determined by PCR amplification from upstream of the 5’ integration site to the 2A element (F: 5’-ATGTTCCACGAAGAAGCCA-3’, R: 5’-TCAGCAGGCTGAAATTGGT-3) followed by qPCR quantification (F: 5’-ATGTTCCACGAAGAAGCCA-3’, R: 5’-AGCTGTTTCTTACTCCATTCTCA-3’, Probe: 5’AGGCAACGTCATGGGTGTGACTTT-3’). A synthesized DNA fragment representing this product was used to generate a standard curve as previously described [28].

2.9. ELISAs.

ALB-2A in plasma was measured by chemiluminescence sandwich ELISA using a proprietary rabbit polyclonal anti-2A antibody for capture and HRP-labelled anti-mouse albumin for detection. FGF21 protein was measured using an ELISA kit (Abcam, ab212160) per the manufacturer’s instructions.

2.10. Statistical analyses.

Prism 8 by GraphPad was used to analyze all data. Results are expressed as mean ± SEM. Values of p<0.05 were considered statistically significant. Depending on the experimental design, an unpaired t-test, log-rank (Mantel-Cox) test, or simple linear regression was used as indicated in the legends of the figures.

3. RESULTS

3.1. Treatment and Survival

Mmut–/–;TgINS-MCK-Mmut (MMA mice) maintained on standard mouse chow were used to study AAV mediated Alb editing. This transgenic mouse model of MMA uses the muscle creatine kinase promoter (MCK) to express a murine Mmut transgene exclusively in the skeletal and cardiac muscle [21]. Mutant mice exhibit increased morbidity and mortality, growth retardation, elevated plasma MMA (pMMA), and elevated plasma FGF21 compared to unaffected, wildtype littermates. The survival of mutant mice is highly dependent on diet and environmental stress. Eleven two-month-old MMA mice received a retro-orbital injection of the AAVDJ-Alb-2A-MMUT vector at a dose of 1e12 vector genomes (VG) per mouse (~7e13 VG/kg). This dose was selected to match the dose used in our previous study in neonates and to facilitate comparisons between studies. The survival of treated and untreated MMA mice is shown in Fig. 1B. Treatment did not significantly increase the survival of the MMA mice (p=0.57; (Mantel-Cox) test). In the treated group (n=11), 3 mice died within days of high stress events, such as a cage flood or following retro-orbital injection, and 1 was sacrificed due to declining health at 4 months post therapy which pathology revealed to be likely unrelated to MMA pathophysiology or AAV treatment. After injection 4 animals were sacrificed at 2 months, and the remaining 3 animals were sacrificed 8 to 10 months post treatment to assess the therapeutic effects.

3.2. Weight and Disease-Associated Plasma Biomarkers

The animals were evaluated monthly for weight gain and disease plasma biomarkers, including plasma methylmalonic acid (pMMA) and FGF21. MMA mice were significantly runted before treatment (mean weight = 12.6g ± 0.8 at 2 months vs. wildtype mean weight = 21.9g ± 0.7 at 2 months) and did not display increased weight gain compared to untreated MMA mice for the duration of the study (p=0.68 at 8 months; unpaired t-test) (Supplemental fig. 1). pMMA was significantly reduced (p<0.05; unpaired t-test) at 2- and 8-months post treatment (4 and 10 months of age) compared to age matched untreated mutant mice (Fig. 1C). At 10 months, treated mutants had an average pMMA of 536μM ± 157 (n=3) compared to age-matched untreated mutants average pMMA of 1150 μM ± 149 (n=7) (p=0.04; unpaired t-test).

Plasma FGF21 levels, hypothesized to reflect mitochondrial dysfunction, are known to be elevated in MMA mice and human patients [21]. Treatment with AAVDJ-Alb-2A-MMUT significantly (p<0.001; unpaired t-test) reduced plasma levels of FGF21 in the juvenile mice (mean = 3.5ng/mL ± 0.78, n=8) compared to untreated mutant mice (mean = 34.8ng/mL ± 5.8, n=5) (Fig. 1D). FGF21 levels were reduced within 1 month of treatment and remained low for the duration of the study (Supplemental Fig 2).

3.3. Hepatic MMUT Expression and Vector Integration

Hepatic MMUT in wildtype control mice, untreated and treated MMA mice was measured by immunoblotting. Protein expression was detected in 2 of 5 MMA animals at 2 to 4 months post treatment (4 animals sacrificed at 2 months and 1 due to declining health at 4 months) and all 3 MMA animals at 8 to 10 months post treatment but not in untreated MMA animals (Fig. 2A). Western blot results were quantified as a percentage of MMUT and normalized to ß-actin expression (Fig. 2B). Treated MMA mice at 8 to 10 months post-treatment had significantly higher levels of MMUT (80% ± 39.1) compared to 2 to 4 months posttreatment (3.4% ± 2.7; p<0.05; unpaired t-test).

Figure 2. Characterization of hepatic editing following treatment with AAVDJ-Alb-2A-MMUT.

Figure 2.

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A. Immunoblot of hepatic MMUT protein (85 kDa) expression in wildtype mice (+ Control), untreated MMA mice (− Control), and treated MMA mice at 2-, 10-, 4- and 8-months post treatment. ß-actin (42 kDa) was used as a loading control. B. Hepatic MMUT protein expression was quantified as a percentage of wildtype MMUT protein expression and normalized to ß-actin expression. (*p<0.05; unpaired t-test) C. Viral copy number represented as the ratio of MMUT genome copies to Gapdh copies as determined by ddPCR. (p=0.28; unpaired t-test) D. The percent of hepatic Alb alleles with MMUT integrations in two distinct liver regions as determined by semi-quantitative PCR. (*p<0.05; unpaired t-test) E. Plasma ALB-2A levels over time in 4 treated MMA mice. F. A simple linear regression of plasma ALB-2A level and the percent of wildtype MMUT protein expression.

Viral copy number in the liver was determined by the ratio of MMUT genome copies to Gapdh copies by ddPCR. There was not a significant difference in the number of viral genomes at 2 to 4 months post treatment (8.9 ± 2.9 genomes) compared to 8 to 10 months post treatment (4.0 ± 2.0 genomes) (p=0.28; unpaired t-test) (Fig. 2C). To determine the integration frequency of MMUT into the Alb locus, we performed a semi-quantitative PCR measurement. Amplification was performed across the recombination site (from the 2A element, through the 5’ arm of homology, to upstream of the 5’ homology arm) and quantified by qPCR as previously described [28] (Supplemental Fig. 3A and 3B). For each mouse, DNA was isolated from two distinct regions of the liver and assayed for integrations. The average of the technical replicates showed that treated MMA mice had significantly more MMUT integrations into the Alb loci at 8 to 10 months post treatment (3.42% ± 2.2) as compared to 2 to 4 months post treatment (0.18% ± 0.32) (p<0.05; unpaired t-test; Fig. 2D and Supplemental fig. 3C).

Corrected hepatocytes produce a 21 amino acid 2A peptide fused to the 3’ terminus of albumin (Fig. 1A), which can be detected in the plasma. When AAVDJ-Alb-2A-MMUT was used to treat MMA mice in the neonatal period, the plasma ALB-2A levels correlated with integration frequency and hepatic protein expression [28]. MMA mice treated as juveniles also have increased levels of plasma ALB-2A, which continue to increase over time (Fig. 2E). The maximum level of plasma ALB-2A detected in each mouse remained a small component of the total albumin produced, with ALB-2A levels never exceeding 3% of the total plasma ALB levels. A linear regression analysis indicated that plasma ALB-2A levels highly correlate with hepatic MMUT protein expression (r2=0.9192, p<0.0005) (Fig. 2F) and were associated with integration events (r2=0.4712, p=0.06).

Livers from treated mice were stained for MMUT mRNA expression and localization by immunohistochemistry. The total number of corrected hepatocytes was quantified by QuPath [36] for each image and demonstrates significant variability between mice the same age post-treatment; however, on average all treated mice show around 20% of hepatocytes were corrected regardless of time post-treatment (Fig. 3A). While the total number of corrected hepatocytes did not vary on average regardless of time post treatment; the pattern of corrected cells was distinct when comparing mice sacrificed shortly after treatment (2 months post treatment, Fig. 3B and 3C) to those sacrificed later (8 months post treatment, Fig. 3D and 10 months post treatment, Fig. 3E). At 2 months post treatment, only isolated individual cells show staining, with no pattern of corrected vs. uncorrected cells (Fig. 3B and 3C). In contrast, at 8- and 10-months post-treatment, corrected cells show a pattern of large, distinct clusters, which is characteristic of clonal expansion of an edited cell (Fig. 3D and 3E). Livers were also stained by H&E and examined by a veterinary pathologist. All examined sections appeared morphologically normal, and no signs of dysplasia or cancer were detected in any of the treated animals.

Figure 3. MMUT mRNA expression in edited hepatocytes.

Figure 3.

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A. The percentage of cells stained for MMUT mRNA in treated mice. B-E. Representative mRNA in situ hybridization images of treated MMA mice. Cells positive for MMUT mRNA are stained brown. The scale bar is 2mm, and the insert (right image) is a 10x magnification. B-C. Representative images from mice 2-months post treatment. D. Representative images from a mouse 8-months post treatment. E. Representative images from a mouse 10-months post treatment.

4. DISCUSSION

MMA is a debilitating multisystemic disorder that remains difficult to manage with the current standard of care. It has been established that MMUT plays an important role in most tissues throughout the body [20], and widespread correction could have a theoretical benefit to treat MMA. However, observations from murine mouse models of MMA with tissue specific expression of MMUT [38] as well as treatment of various murine models of MMA with both constitutive and tissue specific canonical AAV gene therapy vectors [15–18, 39] demonstrate that hepatic expression of MMUT substantially ameliorates disease symptomatology. In addition, observations from patients who receive elective liver transplants demonstrate a reduction of plasma methylmalonic acid levels and a dramatic improvement in metabolic stability [9], providing hope that liver targeted gene therapy with a durable, integrating transgene might deliver similar benefits without the procedural risks associated with transplantation or the need for lifelong immune suppression afterward. We previously demonstrated that AAV vectors utilizing targeted integration into hepatocytes safely and effectively could treat a variety of mouse models of MMA when administered to animals in the neonatal period [28, 40]. In all models, we repeatedly observed a growth advantage conferred upon hepatocytes corrected with targeted, integrating MMUT vectors. In the case of AAVDJ-Alb-2A-MMUT treatment of neonates, this selective advantage allows for the initial, low level of integration to generate a large population of corrected hepatocytes over time. Previous work did not address if this approach would be an effective treatment in older mice or if the treatment of older mice would allow for the selection and expansion of corrected MMA hepatocytes.

Here, we find that clonal expansion of corrected hepatocytes due to selective advantage is variable but present when mice are treated as young adults. Although we had a small number of older treated mice in our study, we consistently observed that MMA mice with the highest levels of plasma ALB-2A also had the highest percentages of Alb integration, more hepatic MMUT production, reduced pMMA, and had more corrected hepatocytes by immunohistochemistry. As seen with neonatal treatment using the same mouse model, the plasma ALB-2A levels correlate with the percentage of Alb integrations, highlighting the utility of this circulating biomarker for longitudinal assessment of editing and in vivo hepatic expansion.

Elevated plasma FGF21 levels in MMA patients have been associated with worsening disease manifestations and correlate with abnormal hepatic mitochondrial ultrastructure [21]. Importantly, MMA patients who have received a liver or combined liver and kidney transplant have dramatically reduced plasma FGF21 levels compared to pretransplant levels. Treatment of MMA mice with a 3’ Alb integrating vector quickly and dramatically reduced plasma FGF21, similar to what has been noted after administration with a canonical AAV8 MMUT vector [21], suggesting that even the very low levels of Alb editing mediated by AAVDJ-Alb-2A-MMUT at early times might mitigate mitochondrial dysfunction.

Treatment of juvenile MMA mice with AAVDJ-Alb-2A-MMUT did not affect mortality, despite pronounced hepatic correction seen at later times. It is essential to highlight that the survival of this strain of MMA mice is highly dependent on diet and environmental stress. In fact, Manoli et al. noted an 87% survival at day of life 120 when mice were kept on a diet containing 21% protein supplemented with carbohydrate rich fruit and Nutrical® and heating strips to reduce temperature fluctuations [21]. However, the same mice exhibit a 43% survival at day 120 of life when maintained on a regular murine diet at ambient temperatures in the setting of standard housing [28]. Because AAVDJ-Alb-2A-MMUT editing relies upon natural HR, which is inefficient, especially in older mice, the mice succumb to the natural history of MMA before an expression benefit of MMUT can be realized. Future efforts should be directed toward either increasing AAV mediated HR using small molecules, such as the ribonucleotide reductase inhibitor fludarabine, or with nuclease enhancement, as demonstrated for Crigler Najjar syndrome. [41, 42].

The results here, and those previously published, support the concept that the underlying hepatopathy of MMA provides an environment that allows a cell autonomous growth advantage to manifest as an edited cell becomes “protected” against the effects of the disease. An increased turnover of uncorrected hepatocytes is also posited, which leads us to speculate that a pool of mitotically active hepatocytes exists to match the demand for hepatocyte turnover in MMA. This pool of replicating hepatocytes would be amenable to AAV mediated HR and perhaps explains the successful editing we document in MMA mice treated as juveniles when liver doubling has ceased. The pattern of MMUT expression in older mice appears as random clusters, which is consistent with clonal proliferation after correction and not with zonulation.

The dosage of AAV used in this study was selected because it is similar to what was administered to neonatal mice in our previous publication (2.5e13 vg/kg to 2.5e14 vg/kg) [28]. However, high doses of AAV are proving to be feasible in an FDA approved therapy for SMA type 1 (1.1e14 vg/kg), and an ongoing phase I/II clinical trial to treat patients with MMA (NCT04581785) using a humanized version of the vector which proposes to treat patients with a dose of 5e13 vg/kg, followed by escalation to 5e14 vg/kg. Dose finding studies in the ongoing phase I/II clinical trial will be critical to define the kinetics of clonal expansion of corrected hepatocytes that might occur in MMA patients after treatment with an analogous, humanized integrating AAV vector [43].

Supplementary Material

1

Acknowledgments

L.E.V., R.J.C., and C.P.V. were supported by the Intramural Research Program of the National Human Genome Research Institute. This work was completed with assistance from the National Cancer Institute Pathology/Histotechnology Laboratory and the CCR Genomics Core. We owe special thanks to Andrew Warner and Elijah Edmondson for their aid in performing RNAscope and analysis. We would also like to acknowledge Darwin Romero for assistance with tracking animals and data entry. This work was funded, in part, by LogicBio Therapeutics, Inc.

FINANCIAL SUPPORT

This work was funded by the Intramural Research Program of the National Human Genome Research Institute and, in part, by LogicBio Therapeutics, Inc.

ABBREVIATIONS

MMA

methylmalonic acidemia

MMUT

methylmalonyl-CoA mutase

AAV

adeno-associated virus

FGF21

fibroblast growth factor 21

Alb

albumin

VG

vector genomes

MCK

muscle creatine kinase promoter

pMMA

plasma methylmalonic acid

Footnotes

Competing Interest Statement

N.R. and J.L. are employees of LogicBio Therapeutics and receive salary support and stock options from LogicBio Therapeutics. S.G. and N.C. are former employees and shareholders of LogicBio Therapeutics.

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