Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 11:4:126-136.
doi: 10.1016/j.omtm.2016.12.010. eCollection 2017 Mar 17.

Low-Dose Liver-Targeted Gene Therapy for Pompe Disease Enhances Therapeutic Efficacy of ERT via Immune Tolerance Induction

Sang-Oh Han  1 , Giuseppe Ronzitti  2 , Benjamin Arnson  1 , Christian Leborgne  2 , Songtao Li  1 , Federico Mingozzi  3 , Dwight Koeberl  4
Affiliations

Low-Dose Liver-Targeted Gene Therapy for Pompe Disease Enhances Therapeutic Efficacy of ERT via Immune Tolerance Induction

Sang-Oh Han et al. Mol Ther Methods Clin Dev. .

Erratum in

Abstract

Pompe disease results from acid α-glucosidase (GAA) deficiency, and enzyme replacement therapy (ERT) with recombinant human (rh) GAA has clinical benefits, although its limitations include the short half-life of GAA and the formation of antibody responses. The present study compared the efficacy of ERT against gene transfer with an adeno-associated viral (AAV) vector containing a liver-specific promoter. GAA knockout (KO) mice were administered either a weekly injection of rhGAA (20 mg/kg) or a single injection of AAV2/8-LSPhGAA (8 × 1011 vector genomes [vg]/kg). Both treatments significantly reduced glycogen content of the heart and diaphragm. Although ERT triggered anti-GAA antibody formation, there was no detectable antibody response following AAV vector administration. The efficacy of three lower dosages of AAV2/8-LSPhGAA was evaluated in GAA-KO mice, either alone or in combination with ERT. The minimum effective dose (MED) identified was 8 × 1010 vg/kg to reduce glycogen content in the heart and diaphragm of GAA-KO mice. A 3-fold higher dose was required to suppress antibody responses to ERT. Efficacy from liver gene therapy was slightly greater in male mice than in female mice. Vector dose correlated inversely with anti-GAA antibody formation, whereas higher vector doses suppressed previously formed anti-GAA antibodies as late as 25 weeks after the start of ERT and achieved biochemical correction of glycogen accumulation. In conclusion, we identified the MED for effective AAV2/8-LSPhGAA-mediated tolerogenic gene therapy in Pompe disease mice.

Keywords: acid maltase; acid α-glucosidase; adeno-associated virus; gene therapy; glycogen storage disease type II; immune tolerance induction.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Comparison of Liver Depot Gene Therapy with ERT In a 5-week study, GAA-KO mice were treated with (A) either a weekly injection of rhGAA (ERT; 20 mg/kg; n = 10) or a single injection of AAV2/8-LSPhGAApA (AAV; 8 × 1011 vg/kg; n = 10). (B–F) The endpoints included: (B) GAA activity and (C) glycogen content in the tissues, (D) antibody formation, (E) blood GAA, and (F) heart size. GAA activity was increased and glycogen content was reduced in the heart and skeletal muscles. Mean ± SD is shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 from ANOVA.
Figure 2
Figure 2
AAV2/8-LSPhGAApA Reduced the Glycogen Content of Muscle and Increased the Benefit from Simultaneous ERT (A) GAA-KO mice were treated with the AAV2/8-LSPhGAA vector (AAV), either with or without simultaneous ERT for four doses (rhGAA, 20 mg/kg every 2 weeks). Number of mice per group was as follows: (−)ERT: 0 (n = 8), 2 × 1010 (n = 8), 8 × 1010 (n = 9), 2 × 1011 (n = 8); (+)ERT: 0 (n = 10), 2 × 1010 (n = 9), 8 × 1010 (n = 9), 2 × 1011 (n = 9). Biochemical correction in GAA-KO mice was evaluated 8 weeks following gene therapy, either with (shaded bars) or without (black bars). GAA activity and glycogen content shown for (B) heart, (C) diaphragm, and (D) quadriceps. Mean ± SD is shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 from two-way ANOVA.
Figure 3
Figure 3
Liver Gene Transfer Reduces IgG Levels in a Dose-Dependent Manner Number of mice per group was as follows: (−)ERT: 0 (n = 8), 2 × 1010 (n = 8), 8 × 1010 (n = 9), 2 × 1011 (n = 8); (+)ERT: 0 (n = 10), 2 × 1010 (n = 9), 8 × 1010 (n = 9), 2 × 1011 (n = 9). (A–D) The histograms indicate the levels of total IgG (A), IgG1 (B), IgG2b (C), and IgG2c (D), measured in plasma 9 weeks after vector injection and 1 week following the fourth dose of ERT. The quantification of antibody isotypes has been performed using purified mouse IgG isotypes as standard. Statistical analysis has been performed by comparison of untreated and ERT-treated mice at the same vector dose using multiple t tests [p < 0.05, (+)ERT versus (−)ERT]. Nine weeks after vector injection mice were sacrificed and the VGCN was measured in the liver. IgG isotype titers were divided in three groups depending on the genome copy number measured in the liver, less than 0.1 (<0.01), between 0.01 and 1 (0.01–1), and more than 1 (>1) copy of vector genome per diploid genome. (E–H) The histograms indicate the levels of total IgG (E), IgG1 (F), IgG2b (G), and IgG2c (H) for each level of VGCN. Statistical analysis has been performed by comparison of untreated and ERT-treated mice at the same vector dose using multiple t tests [p < 0.05, (+)ERT versus (−)ERT]. Mean ± SD is shown. *p < 0.05, ***p < 0.001 from multiple t tests.
Figure 4
Figure 4
Long-Term Clearance of Glycogen from the Heart, Diaphragm, and Skeletal Muscle and Preservation of Neuromuscular Function from Higher Dosage rAAV8 (A) Experimental design. GAA-KO mice were monitored for 36 weeks following administration of ERT with or without a single dose of AAV2/8-LSPhGAA (2 × 1012 vg/kg) at the indicated times. (B) Histograms for anti-GAA ELISA (1:200) detecting IgG1 at the early and late time points. All three groups were challenged with rhGAA at week (Wk) 31. The week 25 administration of rAAV8 suppressed anti-GAA from very elevated at Wk 23 to background levels by week 36. However, the ERT-only group formed higher antibodies following the immune challenge. AAV at week 5 maintained suppression of anti-GAA. The background signal for untreated mice was <0.01 in the ELISA. (C) Rotarod at week 0 (baseline) and week 36. Mice were euthanized at week 36. (D and E) Muscle GAA (D) and muscle glycogen (E). Mean ± SD is shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, per two-way ANOVA.
Figure 5
Figure 5
Liver Gene Transfer Eradicates Humoral Immune Response Triggered by Multiple rhGAA Infusions GAA-KO mice from the three groups were injected with 20 mg/kg rhGAA and 8 × 1011 vg/kg AAV8-LSPhGAA as indicated in the experimental plan (Figure 4A). Scatterplots show total IgG (A), IgG1 (B), and ranked IgG (C) and IgG1 (D), which was measured in plasma 36 weeks after vector injection and ranked by the degree of antibody production. Antibodies were ranked as follows: <0.25 = 0, 0.25–0.5 = 1, 0.51–0.75 = 2, 0.76–1 = 3, and >1 = 4 (Table S1). The quantification of antibody isotypes has been performed using purified mouse IgG isotypes as standard. Mean ± SD is shown. *p < 0.05, **p < 0.01 from ANOVA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hirschhorn R., Reuser A.J.J. Chapter 135. In: Scriver C.R., Beaudet A.L., Sly W.S., Valle D., editors. The Metabolic and Molecular Basis for Inherited Disease. Eighth Edition. McGraw-Hill; 2001. pp. 3389–3419.
    1. Banugaria S.G., Prater S.N., Ng Y.K., Kobori J.A., Finkel R.S., Ladda R.L., Chen Y.T., Rosenberg A.S., Kishnani P.S. The impact of antibodies on clinical outcomes in diseases treated with therapeutic protein: lessons learned from infantile Pompe disease. Genet. Med. 2011;13:729–736. - PMC - PubMed
    1. Messinger Y.H., Mendelsohn N.J., Rhead W., Dimmock D., Hershkovitz E., Champion M., Jones S.A., Olson R., White A., Wells C. Successful immune tolerance induction to enzyme replacement therapy in CRIM-negative infantile Pompe disease. Genet. Med. 2012;14:135–142. - PMC - PubMed
    1. Banugaria S.G., Prater S.N., Patel T.T., Dearmey S.M., Milleson C., Sheets K.B., Bali D.S., Rehder C.W., Raiman J.A., Wang R.A. Algorithm for the early diagnosis and treatment of patients with cross reactive immunologic material-negative classic infantile pompe disease: a step towards improving the efficacy of ERT. PLoS ONE. 2013;8:e67052. - PMC - PubMed
    1. van der Ploeg A.T., Clemens P.R., Corzo D., Escolar D.M., Florence J., Groeneveld G.J., Herson S., Kishnani P.S., Laforet P., Lake S.L. A randomized study of alglucosidase alfa in late-onset Pompe’s disease. N. Engl. J. Med. 2010;362:1396–1406. - PubMed