TY - JOUR
T1 - Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease
AU - Heller, Gregory J.
AU - Marshall, Michael S.
AU - Issa, Yazan
AU - Marshall, Jeffrey N.
AU - Nguyen, Duc
AU - Rue, Emily
AU - Pathmasiri, Koralege C.
AU - Domowicz, Miriam S.
AU - van Breemen, Richard B.
AU - Tai, Leon M.
AU - Cologna, Stephanie M.
AU - Crocker, Stephen J.
AU - Givogri, Maria I.
AU - Sands, Mark S.
AU - Bongarzone, Ernesto R.
N1 - Funding Information:
We thank Dr Steve Gray for the generous gift of the AAV-001-GFP vector and the technical assistance of Benas Jakobauskas, Monika Stoskute, and Alexandra Socovich. M.S.M. was funded through a pre-doctoral NRSA fellowship from the NIH (NRSA F30NS090684). E.R.B. was funded with grants from the NIH (R01 NS065808), the Legacy of Angels Foundation, Partners for Krabbe Disease Research, and the European Leukodystrophy Association. G.J.H. M.S.M. and E.R.B. designed the study and analyzed the data; G.J.H. and M.S.M. administered all of the treatments; M.S.M. and D.N. took care of the mouse colony; G.J.H. M.S.M. Y.I. and J.N.M. collected the tissue; M.S.M. prepared the psychosine extractions; K.C.P. S.M.C. E.R. and R.B.v.B. performed the MS measurements and the analysis of the psychosine concentrations; G.J.H. M.S.M. Y.I. J.N.M. L.M.T. and E.R.B. performed all of the histological staining and confocal and epifluorescent microscopy imaging; E.R.B. performed the EM imaging and analysis; M.I.G. and S.J.C. performed the immunoblotting; M.S.S. performed the independent quality control experiments for lesions at Washington University in St. Louis; G.J.H. cultured the neuroglial cells and performed qPCR as well as quantified the GALC activity on the lysates; G.J.H. M.S.M. and E.R.B. prepared the figures; G.J.H. M.S.M. and E.R.B. wrote the manuscript, with contributions from L.M.T. S.M.C. S.J.C. M.S.S. and M.I.G. All of the authors read and approved the manuscript. E.R.B. is a consultant for Lysosomal Therapeutics, E-Scape Bio, Gain Therapeutics, Affinia Therapeutics, and Neurogene. None of these entities participated in or contributed financially or intellectually to this work and the decision to publish.
Funding Information:
We thank Dr Steve Gray for the generous gift of the AAV-001-GFP vector and the technical assistance of Benas Jakobauskas, Monika Stoskute, and Alexandra Socovich. M.S.M. was funded through a pre-doctoral NRSA fellowship from the NIH (NRSA F30NS090684 ). E.R.B. was funded with grants from the NIH ( R01 NS065808 ), the Legacy of Angels Foundation , Partners for Krabbe Disease Research , and the European Leukodystrophy Association .
Publisher Copyright:
© 2021 The Author(s)
PY - 2021/5/5
Y1 - 2021/5/5
N2 - Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6–8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.
AB - Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6–8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.
KW - AAV9
KW - demyelination
KW - fibrinogen
KW - gene therapy
KW - globoid cell leukodystrophy
KW - lysosomal dysfunction
KW - microglia
KW - multi-focal sclerosis
KW - psychosine
KW - tight junction
UR - http://www.scopus.com/inward/record.url?scp=85100557654&partnerID=8YFLogxK
U2 - 10.1016/j.ymthe.2021.01.026
DO - 10.1016/j.ymthe.2021.01.026
M3 - Article
C2 - 33508430
AN - SCOPUS:85100557654
SN - 1525-0016
VL - 29
SP - 1883
EP - 1902
JO - Molecular Therapy
JF - Molecular Therapy
IS - 5
ER -