A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency

Olav M. Andersen; Nikolaj Bøgh; Anne M. Landau; Gro G. Pløen; Anne Mette G. Jensen; Giulia Monti; Benedicte P. Ulhøi; Jens R. Nyengaard; Kirsten R. Jacobsen; Margarita M. Jørgensen; Ida E. Holm; Marianne L. Kristensen; Aage Kristian O. Alstrup; Esben S. S. Hansen; Charlotte E. Teunissen; Laura Breidenbach; Mathias Droescher; Ying Liu; Hanne S. Pedersen; Henrik Callesen; Yonglun Luo; Lars Bolund; David J. Brooks; Christoffer Laustsen; Scott A. Small; Lars F. Mikkelsen; Charlotte B. Sørensen

doi:https://doi.org/10.1016/j.xcrm.2022.100740

A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency

Olav M. Andersen, Nikolaj Bøgh, Anne M. Landau, Gro G. Pløen, Anne Mette G. Jensen, Giulia Monti, Benedicte P. Ulhøi, Jens R. Nyengaard, Kirsten R. Jacobsen, Margarita M. Jørgensen, Ida E. Holm, Marianne L. Kristensen, Aage Kristian O. Alstrup, Esben S. S. Hansen, Charlotte E. Teunissen, Laura Breidenbach, Mathias Droescher, Ying Liu, Hanne S. Pedersen, Henrik CallesenYonglun Luo, Lars Bolund, David J. Brooks, Christoffer Laustsen, Scott A. Small, Lars F. Mikkelsen, Charlotte B. Sørensen

Research output: Contribution to journal › Article › Academic › peer-review

18 Citations (Scopus)

Abstract

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%–3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of β-amyloid (Aβ) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

Original language	English
Article number	100740
Journal	Cell Reports Medicine
Volume	3
Issue number	9
DOIs	https://doi.org/10.1016/j.xcrm.2022.100740
Publication status	Published - 20 Sept 2022

Keywords

Alzheimer's disease
CRISPR-Cas9
SORL1
SORLA
genome editing
large animal model
retromer-dependent endosomal recycling

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https://doi.org/10.1016/j.xcrm.2022.100740

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Andersen, O. M., Bøgh, N., Landau, A. M., Pløen, G. G., Jensen, A. M. G., Monti, G., Ulhøi, B. P., Nyengaard, J. R., Jacobsen, K. R., Jørgensen, M. M., Holm, I. E., Kristensen, M. L., Alstrup, A. K. O., Hansen, E. S. S., Teunissen, C. E., Breidenbach, L., Droescher, M., Liu, Y., Pedersen, H. S., ... Sørensen, C. B. (2022). A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency. Cell Reports Medicine, 3(9), Article 100740. https://doi.org/10.1016/j.xcrm.2022.100740

@article{a256ecd6e4834fdcbb77c2fa5e64374f,

title = "A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency",

abstract = "The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%–3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in G{\"o}ttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of β-amyloid (Aβ) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.",

keywords = "Alzheimer's disease, CRISPR-Cas9, SORL1, SORLA, genome editing, large animal model, retromer-dependent endosomal recycling",

author = "Andersen, {Olav M.} and Nikolaj B{\o}gh and Landau, {Anne M.} and Pl{\o}en, {Gro G.} and Jensen, {Anne Mette G.} and Giulia Monti and Ulh{\o}i, {Benedicte P.} and Nyengaard, {Jens R.} and Jacobsen, {Kirsten R.} and J{\o}rgensen, {Margarita M.} and Holm, {Ida E.} and Kristensen, {Marianne L.} and Alstrup, {Aage Kristian O.} and Hansen, {Esben S. S.} and Teunissen, {Charlotte E.} and Laura Breidenbach and Mathias Droescher and Ying Liu and Pedersen, {Hanne S.} and Henrik Callesen and Yonglun Luo and Lars Bolund and Brooks, {David J.} and Christoffer Laustsen and Small, {Scott A.} and Mikkelsen, {Lars F.} and S{\o}rensen, {Charlotte B.}",

note = "Funding Information: We thank Kjeld Dahl Winther (SEGES, Denmark) for excellent veterinary assistance during CSF sampling. We are also grateful to Bert Vogelstein and Kenneth W. Kinzler (Johns Hopkins University, Baltimore, MD) for kindly providing the pNeDaKO plasmid vector. We also thank Trine S. Petersen, Lisa Maria R{\o}ge, Dorte Qualmann, Anette M. Pedersen, Janne Adamsen, Klaus Villemoes, Adrian Zeltner, Michelle S{\o}rensen, Sandra Bonnesen, and Benedicte Vestergaard for skilled technical assistance and Anne Mette V. Toft, Mette Bak, and Martin A. Fredsted for skillful assistance with animal handling and transport to scanning facilities. We are grateful to Asad Jan and Ken P. Kragsfeldt for help with microscopy analysis and illustrations, respectively. This study was carried out in accordance to the ARRIVE guidelines and was supported by grants to C.B.S. from the Lundbeck Foundation (R100-A9209), The Danish Heart Association (16-R107-A6813-22997), the Ellegaard G{\"o}ttingen Minipigs Research Foundation, and Ellegaard G{\"o}ttingen Minipigs A/S. O.M.A. performed initial cloning of targeting constructs, conducted analysis on CSF, collected animal samples, and performed WB experiments, preparation of illustrations, and interpretation of data. N.B. A.K.O.A. E.S.S.H. and C.L. performed MRI analyses. A.M.L. A.K.O.A. and D.J.B. conducted PET analyses. G.G.P. genotyped animals, performed CRISPR off-target analysis, and established primary fibroblast cultures from G{\"o}ttingen minipig ear biopsies. A.M.G.J. performed MSD assays for CSF samples and IHC for brain tissue. G.M. performed RT-PCR, WB analysis for brain homogenates, and ICC analysis on cultured fibroblasts. B.P.U. performed neuropathological analysis of the 5-month-old animals. J.R.N. consulted on the neuropathological analysis. K.R.J. supervised selection of control G{\"o}ttingen minipigs and control tissue to include in the study. M.M.J. and I.E.H. dissected minipig brains and performed neuropathological analysis for amyloid plaques and fibrillary tangles. M.L.K. performed IHC on brain tissue. C.E.T. consulted on CSF AD biomarkers. L.B. and M.D. conducted the neurofilament light analyses on CSF. Y.L. performed manual cloning and culturing of the reconstructed embryos. H.S.P. and H.C. performed embryo transfer and supervised delivery and monitoring of the cloned piglets. Y.L. provided vectors for construction of sgRNA and C-check plasmids. L.B. consulted on the initial study design. S.A.S. consulted on data interpretation. L.F.M. provided G{\"o}ttingen minipig fibroblasts for genetic modification and control animals for the study. C.B.S. constructed sgRNA vectors; generated the genetically modified donor cells for cloning; conducted southern blot analyses, animal sampling, genotyping, RT-PCR, and qPCR analyses on brain tissues; prepared illustrations; and interpreted data. O.M.A. and C.B.S. conceptualized and supervised the entire study and wrote the manuscript together with S.A.S. L.B. and M.D. are employees of AbbVie and own AbbVie stock. AbbVie participated in the design and study conduct for this research as well as in the interpretation of data, review, and approval of the publication. Ellegaard G{\"o}ttingen Minipigs A/S has the commercialization rights to the genetically altered G{\"o}ttingen minipig SORL1 model. S.A.S. is a co-founder of Retromer Therapeutics, has equity in the company, and is a paid consultant to the company. O.M.A. also has commercial interests in Retromer Therapeutics, but the company was not involved in any aspects of the study. Funding Information: We thank Kjeld Dahl Winther (SEGES, Denmark) for excellent veterinary assistance during CSF sampling. We are also grateful to Bert Vogelstein and Kenneth W. Kinzler (Johns Hopkins University, Baltimore, MD) for kindly providing the pNeDaKO plasmid vector. We also thank Trine S. Petersen, Lisa Maria R{\o}ge, Dorte Qualmann, Anette M. Pedersen, Janne Adamsen, Klaus Villemoes, Adrian Zeltner, Michelle S{\o}rensen, Sandra Bonnesen, and Benedicte Vestergaard for skilled technical assistance and Anne Mette V. Toft, Mette Bak, and Martin A. Fredsted for skillful assistance with animal handling and transport to scanning facilities. We are grateful to Asad Jan and Ken P. Kragsfeldt for help with microscopy analysis and illustrations, respectively. This study was carried out in accordance to the ARRIVE guidelines and was supported by grants to C.B.S. from the Lundbeck Foundation ( R100-A9209 ), The Danish Heart Association ( 16-R107-A6813-22997 ), the Ellegaard G{\"o}ttingen Minipigs Research Foundation , and Ellegaard G{\"o}ttingen Minipigs A/S . Publisher Copyright: {\textcopyright} 2022 The Author(s)",

year = "2022",

month = sep,

day = "20",

doi = "https://doi.org/10.1016/j.xcrm.2022.100740",

language = "English",

volume = "3",

journal = "Cell Reports Medicine",

issn = "2666-3791",

publisher = "Cell Press",

number = "9",

}

Andersen, OM, Bøgh, N, Landau, AM, Pløen, GG, Jensen, AMG, Monti, G, Ulhøi, BP, Nyengaard, JR, Jacobsen, KR, Jørgensen, MM, Holm, IE, Kristensen, ML, Alstrup, AKO, Hansen, ESS, Teunissen, CE, Breidenbach, L, Droescher, M, Liu, Y, Pedersen, HS, Callesen, H, Luo, Y, Bolund, L, Brooks, DJ, Laustsen, C, Small, SA, Mikkelsen, LF & Sørensen, CB 2022, 'A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency', Cell Reports Medicine, vol. 3, no. 9, 100740. https://doi.org/10.1016/j.xcrm.2022.100740

TY - JOUR

T1 - A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency

AU - Andersen, Olav M.

AU - Bøgh, Nikolaj

AU - Landau, Anne M.

AU - Pløen, Gro G.

AU - Jensen, Anne Mette G.

AU - Monti, Giulia

AU - Ulhøi, Benedicte P.

AU - Nyengaard, Jens R.

AU - Jacobsen, Kirsten R.

AU - Jørgensen, Margarita M.

AU - Holm, Ida E.

AU - Kristensen, Marianne L.

AU - Alstrup, Aage Kristian O.

AU - Hansen, Esben S. S.

AU - Teunissen, Charlotte E.

AU - Breidenbach, Laura

AU - Droescher, Mathias

AU - Liu, Ying

AU - Pedersen, Hanne S.

AU - Callesen, Henrik

AU - Luo, Yonglun

AU - Bolund, Lars

AU - Brooks, David J.

AU - Laustsen, Christoffer

AU - Small, Scott A.

AU - Mikkelsen, Lars F.

AU - Sørensen, Charlotte B.

N1 - Funding Information: We thank Kjeld Dahl Winther (SEGES, Denmark) for excellent veterinary assistance during CSF sampling. We are also grateful to Bert Vogelstein and Kenneth W. Kinzler (Johns Hopkins University, Baltimore, MD) for kindly providing the pNeDaKO plasmid vector. We also thank Trine S. Petersen, Lisa Maria Røge, Dorte Qualmann, Anette M. Pedersen, Janne Adamsen, Klaus Villemoes, Adrian Zeltner, Michelle Sørensen, Sandra Bonnesen, and Benedicte Vestergaard for skilled technical assistance and Anne Mette V. Toft, Mette Bak, and Martin A. Fredsted for skillful assistance with animal handling and transport to scanning facilities. We are grateful to Asad Jan and Ken P. Kragsfeldt for help with microscopy analysis and illustrations, respectively. This study was carried out in accordance to the ARRIVE guidelines and was supported by grants to C.B.S. from the Lundbeck Foundation (R100-A9209), The Danish Heart Association (16-R107-A6813-22997), the Ellegaard Göttingen Minipigs Research Foundation, and Ellegaard Göttingen Minipigs A/S. O.M.A. performed initial cloning of targeting constructs, conducted analysis on CSF, collected animal samples, and performed WB experiments, preparation of illustrations, and interpretation of data. N.B. A.K.O.A. E.S.S.H. and C.L. performed MRI analyses. A.M.L. A.K.O.A. and D.J.B. conducted PET analyses. G.G.P. genotyped animals, performed CRISPR off-target analysis, and established primary fibroblast cultures from Göttingen minipig ear biopsies. A.M.G.J. performed MSD assays for CSF samples and IHC for brain tissue. G.M. performed RT-PCR, WB analysis for brain homogenates, and ICC analysis on cultured fibroblasts. B.P.U. performed neuropathological analysis of the 5-month-old animals. J.R.N. consulted on the neuropathological analysis. K.R.J. supervised selection of control Göttingen minipigs and control tissue to include in the study. M.M.J. and I.E.H. dissected minipig brains and performed neuropathological analysis for amyloid plaques and fibrillary tangles. M.L.K. performed IHC on brain tissue. C.E.T. consulted on CSF AD biomarkers. L.B. and M.D. conducted the neurofilament light analyses on CSF. Y.L. performed manual cloning and culturing of the reconstructed embryos. H.S.P. and H.C. performed embryo transfer and supervised delivery and monitoring of the cloned piglets. Y.L. provided vectors for construction of sgRNA and C-check plasmids. L.B. consulted on the initial study design. S.A.S. consulted on data interpretation. L.F.M. provided Göttingen minipig fibroblasts for genetic modification and control animals for the study. C.B.S. constructed sgRNA vectors; generated the genetically modified donor cells for cloning; conducted southern blot analyses, animal sampling, genotyping, RT-PCR, and qPCR analyses on brain tissues; prepared illustrations; and interpreted data. O.M.A. and C.B.S. conceptualized and supervised the entire study and wrote the manuscript together with S.A.S. L.B. and M.D. are employees of AbbVie and own AbbVie stock. AbbVie participated in the design and study conduct for this research as well as in the interpretation of data, review, and approval of the publication. Ellegaard Göttingen Minipigs A/S has the commercialization rights to the genetically altered Göttingen minipig SORL1 model. S.A.S. is a co-founder of Retromer Therapeutics, has equity in the company, and is a paid consultant to the company. O.M.A. also has commercial interests in Retromer Therapeutics, but the company was not involved in any aspects of the study. Funding Information: We thank Kjeld Dahl Winther (SEGES, Denmark) for excellent veterinary assistance during CSF sampling. We are also grateful to Bert Vogelstein and Kenneth W. Kinzler (Johns Hopkins University, Baltimore, MD) for kindly providing the pNeDaKO plasmid vector. We also thank Trine S. Petersen, Lisa Maria Røge, Dorte Qualmann, Anette M. Pedersen, Janne Adamsen, Klaus Villemoes, Adrian Zeltner, Michelle Sørensen, Sandra Bonnesen, and Benedicte Vestergaard for skilled technical assistance and Anne Mette V. Toft, Mette Bak, and Martin A. Fredsted for skillful assistance with animal handling and transport to scanning facilities. We are grateful to Asad Jan and Ken P. Kragsfeldt for help with microscopy analysis and illustrations, respectively. This study was carried out in accordance to the ARRIVE guidelines and was supported by grants to C.B.S. from the Lundbeck Foundation ( R100-A9209 ), The Danish Heart Association ( 16-R107-A6813-22997 ), the Ellegaard Göttingen Minipigs Research Foundation , and Ellegaard Göttingen Minipigs A/S . Publisher Copyright: © 2022 The Author(s)

PY - 2022/9/20

Y1 - 2022/9/20

N2 - The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%–3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of β-amyloid (Aβ) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

AB - The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%–3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of β-amyloid (Aβ) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

KW - Alzheimer's disease

KW - CRISPR-Cas9

KW - SORL1

KW - SORLA

KW - genome editing

KW - large animal model

KW - retromer-dependent endosomal recycling

UR - http://www.scopus.com/inward/record.url?scp=85138210540&partnerID=8YFLogxK

U2 - https://doi.org/10.1016/j.xcrm.2022.100740

DO - https://doi.org/10.1016/j.xcrm.2022.100740

M3 - Article

C2 - 36099918

SN - 2666-3791

VL - 3

JO - Cell Reports Medicine

JF - Cell Reports Medicine

IS - 9

M1 - 100740

ER -

A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency

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