Antisense oligonucleotides targeting the miR-29b binding site in the GRN mRNA increase progranulin translation

Abstract

Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3′ UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed for miR-29b from binding to the GRN 3′ UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by polysome profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency. Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3′ UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed for miR-29b from binding to the GRN 3′ UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by polysome profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency. Progranulin is a lysosomal and secreted protein with pleiotropic effects, including promoting neuronal survival, neurite outgrowth, wound healing, tumor cell growth, and modulating inflammation (1Kao A.W. McKay A. Singh P.P. Brunet A. Huang E.J. 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AlzForum Foundation Inc, Boston, MA2021Google Scholar), and protein replacement (21Logan T. Simon M.J. Rana A. Cherf G.M. Srivastava A. Davis S.S. et al.Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.Cell. 2021; 184: 4651-4668.e4625Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, there are currently no approved therapies for progranulin-deficient FTD. Antisense oligonucleotides (ASOs), short oligonucleotides used to modulate target are emerging as a promising therapeutic modality for neurological Miller Antisense from mouse models to human neurodegenerative 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. Antisense oligonucleotides for 2020; PubMed Scopus Google Scholar). The used ASO strategies of the target and of of the target Miller Antisense from mouse models to human neurodegenerative 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). strategies for increasing target protein levels are limited. Several strategies including and in the UTR W. H. of is increased by oligonucleotides 2016; PubMed Scopus Google Scholar, H. W. S. J. et oligonucleotides in can increase protein 2017; PubMed Scopus Google Scholar). However, these are by the that to regulate of human are of 2009; PubMed Scopus Google Scholar). to the 3′ UTR of target and protein levels Y. The of and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. H. Y. C. of of and 2018; PubMed Scopus Google Scholar). progranulin, to negatively regulate progranulin protein miR-29b, and J. the expression of human progranulin, a secreted in frontotemporal Scopus Google Scholar, L. Q. J. S. Zheng H. J. et and the and of by Mol. 2017; PubMed Scopus Google Scholar, R. Baker M. T. Z. 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S.J. et in the of GRN is a major risk factor for frontotemporal Mol. 2008; PubMed Scopus Google Scholar), is a risk factor for FTD R. Baker M. T. Z. S.J. et in the of GRN is a major risk factor for frontotemporal Mol. 2008; PubMed Scopus Google Scholar) and Alzheimer’s disease J. Su L. Xu Z. Chen G. Progranulin polymorphism rs5848 is associated with increased risk of Alzheimer’s disease.Gene. 2014; 542: 141-145Crossref PubMed Scopus (46) Google Scholar, M.J. Chen M.J. rs5848 of progranulin gene is a risk of Alzheimer’s disease in the 2011; PubMed Scopus Google Scholar). In predict that the binding of to the GRN with the a binding and with with the rs5848 progranulin protein in brain and in R. Baker M. T. Z. S.J. et in the of GRN is a major risk factor for frontotemporal Mol. 2008; PubMed Scopus Google Scholar, H. G. van R. et al.Progranulin levels in plasma and in mutation 2016; PubMed Scopus Google Scholar), that of progranulin levels contributes to the risk of these forms of these as potential for increasing progranulin In the we tested a therapeutic strategy for increasing progranulin protein levels by ASOs to target the miR-29b binding site in the human GRN mRNA. We that these ASOs effectively increase progranulin protein in in iPSC-derived and in mouse we the of is that these ASOs miR-29b from its binding site and translation, in increased of progranulin studies that of miR-29b progranulin protein levels in J. the expression of human progranulin, a secreted in frontotemporal Scopus Google Scholar, L. Q. J. S. Zheng H. J. et and the and of by Mol. 2017; PubMed Scopus Google Scholar). We that treatment with a miR-29b inhibitor progranulin protein levels in human and in human neuroglioma expression of miR-29b from the N. P. K. C. T. C. et of expression human 2016; PubMed Scopus Google Scholar) that miR-29b is in the human brain of miR-29b levels of other miR-29b and other A.J. Y. Y. X. M. The cell and to and PubMed Scopus Google Scholar), we to ASOs the miR-29b binding site in the GRN can increase progranulin protein levels in a we a of ASOs with and that the miR-29b binding site with and in ASOs that increased progranulin protein to a ASO We these ASOs and ASOs that increased progranulin protein levels as by these we a subset of these ASOs against a of ASOs with and found that the of these the results that the of the ASOs are the of progranulin is also secreted from we tested the ASOs increased progranulin in the We found that ASOs that increased progranulin levels also increased secreted progranulin levels that the in progranulin levels are due to trafficking We that the ASOs in with the and the of the miR-29b binding site ASOs with the binding The of the GRN that forms a increase for ASO is in to the miR-29b binding site and may with the miR-29b binding in the efficacy of ASOs we to on and we and for ASOs in cells. The studies revealed that increased progranulin protein levels are of treatment for ASO and by for ASO The of ASO may be due its binding as it a ASO the of these ASO is with of of ASOs by that is E. P. Zhang P. S. M. E. et and of trafficking of oligonucleotides human cell PubMed Scopus Google Scholar) and of of a to A. M. A. S. A of 2013; PubMed Scopus Google Scholar). that the ASOs dose-dependent The of these ASOs to as by for for for ASOs also increase progranulin levels in a dose-dependent manner in iPSC-derived neurons ASOs no in as by We tested the ASOs miR-29b by a miR-29b This inhibitor to miR-29b and miR-29b binding to its target The of demonstrate that ASOs and increased progranulin levels to the miR-29b the to increase progranulin levels in the of the miR-29b that the ASOs on ASO with the miR-29b inhibitor increase progranulin due to the of ASO to ASOs and the ASOs can for binding of miR-29b, we used a FRET-based to the in miR-29b and a GRN 3′ UTR that the miR-29b binding we that ASO effectively competed miR-29b with of In the ASO to Together, these that the miR-29b and for miR-29b binding to the GRN mRNA. increasing progranulin protein the ASOs increase GRN levels in to a ASO that the ASOs increase the of progranulin This be with the of by Y. The of and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. H. Y. C. of of and 2018; PubMed Scopus Google Scholar). we polysome to the of GRN its of We found that with ASOs and of GRN in the K. Brouwers N. Van Damme P. Engelborghs S. Gijselinck I. van der Zee J. et al.Serum biomarker for progranulin-associated frontotemporal lobar degeneration.Ann. Neurol. 2009; 65: 603-609Crossref PubMed Scopus (179) Google Scholar, J. Verfaillie C.M. Van Damme P. Tweaking progranulin expression: therapeutic avenues and opportunities.Front. Mol. Neurosci. 2021; 14713031Crossref PubMed Scopus (21) Google Scholar, 13Amin S. Carling G. Gan L. New insights and therapeutic opportunities for progranulin-deficient frontotemporal dementia.Curr. Opin. Neurobiol. 2021; 72: 131-139Crossref PubMed Scopus (11) Google Scholar, A.E. Filiano A.J. Unger D.E. Young A.H. Roberson E.D. Restoring neuronal progranulin reverses deficits in a mouse model of frontotemporal dementia.Brain. 2017; 140: 1447-1465Crossref PubMed Scopus (49) Google Scholar, B. Sephton C. Dewey C. Xian X. Wei S. Yu K. et al.Suberoylanilide hydroxamic acid (vorinostat) up-regulates progranulin transcription: rational therapeutic approach to frontotemporal dementia.J. Biol. Chem. 2011; 286: 16101-16108Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 16Holler C.J. Taylor G. McEachin Z.T. Deng Q. Watkins W.J. Hudson K. et al.Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: a novel therapeutic lead to treat frontotemporal dementia.Mol. Neurodegener. 2016; 11: 46Crossref PubMed Scopus (76) Google Scholar), to with a ASO that these ASOs increase the of progranulin This to GRN as it with is linked to and FTD. the ASOs increase the of newly synthesized progranulin we used a to D.C. A.J. J. identification of newly synthesized in acid Sci. S. A. 2006; PubMed Scopus Google Scholar). in we with ASOs for from the by the of for for the cell we a to to of progranulin, that ASOs and increase the of progranulin protein Together, these studies the of is that these ASOs miR-29b from its binding site and translation, in increased of progranulin we tested ASO in a humanized GRN mouse model B. G. J. W. et mice as a novel for the of 2021; PubMed Scopus Google Scholar). This model is a mouse in the human GRN gene the 3′ in the on the mouse of ASO P. B. A. H. J. et of and in the of and 2021; PubMed Scopus Google Scholar), we increased human progranulin protein by in the of mice that ASO Progranulin levels increased by and in and to mice of the that the we also increased human progranulin levels in the and of mice that ASO are with the ASO in these brain as by that the ASO Miller A.J. et neuronal loss and reverses and in mice with Transl. 2017; PubMed Scopus Google Scholar). ASO increase GRN levels in the that it progranulin translation, to our in of inflammation in mice that ASO and the ASO ASO in mice tested a on and of inflammation in the Together, these results demonstrate that ASOs the miR-29b binding site can increase human progranulin protein levels in and in In the we that ASOs the miR-29b binding site can increase human progranulin protein levels in in iPSC-derived and in the of humanized GRN In the ASO are dose-dependent and are studies revealed that the ASOs miR-29b, for miR-29b binding to the 3′ UTR of the GRN increase the of progranulin translation, and increase the of newly synthesized progranulin our is the of ASOs to increase the of a target protein by sterically blocking a miR binding Several studies used oligonucleotides to miR binding A.J. and of and by PubMed Scopus Google Scholar, S. Y. N. B. negatively PubMed Scopus Google Scholar, D. T. J. M. T. et expression: for 2011; PubMed Scopus Google Scholar, H. A.J. regulation of to cell 2011; PubMed Scopus Google Scholar, A.J. of target to the of in 2011; PubMed Scopus Google Scholar, A. J. J. R. et factors and that regulate to expression are for J. PubMed Scopus Google Scholar), but to our is the strategy to increase progranulin levels. This strategy The increased progranulin protein is from the GRN and is to and from GRN expression forms of progranulin used in other therapeutic strategy progranulin protein levels a and from of progranulin, as W. G. factor and expression in PubMed Scopus Google Scholar, Chen X. S. et and of PubMed Scopus Google Scholar). by partially with the miR-29b binding site in the GRN the of our approach by other miR-29b target due to in the the binding ASO strategy the miR-29b binding site is to the disease mutation and be used in the of of the GRN mutations that M. J. Van C. mutation for neurodegenerative brain PubMed Scopus Google Scholar). Several of that the in progranulin protein levels treatment with the ASOs are the of by sterically blocking miR-29b binding to its binding site in the GRN 3′ the ASOs are to for miR-29b binding to the GRN 3′ UTR in a FRET-based the are on miR-29b, as the ASOs increase progranulin protein levels miR-29b is the in the progranulin levels with the ASOs in and in are to the with a miR-29b inhibitor in polysome the ASOs increase the of GRN with is linked to and FTD. in found no in the human with for of the ASOs used in for revealed a of potential are in the human brain on the expression 2013; PubMed Scopus Google Scholar), and of the potential are by on progranulin protein levels with ASOs in to ASOs and ASOs with and the case that these are is ASOs increase progranulin with the binding on the of the GRN the by ASOs a provide increased for ASO is in to the miR-29b binding site and may with the miR-29b binding in the efficacy of ASOs to ASOs that with the binding ASO progranulin and in a manner ASOs are to be a promising therapeutic modality for diseases, as ASOs are of well in humans, and in the with a of Miller Antisense from mouse models to human neurodegenerative 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. Antisense oligonucleotides for 2020; PubMed Scopus Google Scholar). studies on these ASOs the miR-29b binding site can FTD-associated neuropathology and behavioral deficits in mouse the study on ASOs that target the miR-29b binding we also tested a of ASOs the binding site R. Baker M. T. Z. S.J. et in the of GRN is a major risk factor for frontotemporal Mol. 2008; PubMed Scopus Google Scholar, P. M. F. A. M. et levels with progranulin increase in for frontotemporal Mol. Neurosci. 2016; PubMed Scopus (21) Google Scholar) in the GRN 3′ UTR and ASOs that increase progranulin protein levels ASOs the and miR-29b binding may provide to increase progranulin in results that ASO strategy may be for increasing levels of target and be for the of therapies for of haploinsufficiency. ASOs used in these studies ASOs with with a with the ASOs used in the in with with a ASO in with with a and the miR-29b inhibitor is a ASO and ASO and are in and ASOs used for in and studies in and in ASOs in and by a The from miR-29b inhibitor and with with human human neuroglioma and human from in with and of in with and of cell from neuronal in with and acid for with other ASO in and with ASOs as on the of miR-29b and expression for neurons by and as M.S. R. C. C. Gan L. of human Biol. 2018; PubMed Scopus Google Scholar). of well in neurons with ASOs for to ASO by and and progranulin with and in to and protein the progranulin in to of well of protein a secreted progranulin, and for to the and the for of protein of on to the blocking and with and a antibodies used for progranulin that to B. Yu G. J. et progranulin is a and is a of Biol. Chem. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar), monoclonal and monoclonal The antibodies used and from the with to the and with a for human as human for to the gene and by the of miR the small from of the the and on the to the human and are as on miR expression levels in human from the N. P. K. C. T. C. et of expression human 2016; PubMed Scopus Google Scholar). The of the human GRN the M. for acid and PubMed Scopus Google Scholar). in and with ASOs for to to the cell a of with and in In and in and the the and on a to for in a a of of and of with and a and in a of and the in to and by to and for The to a of and of for with and for The and in of with for of for with a a as human human human and human levels a with The is to the of the the polysome from the in with in and with ASOs for a of of the acid for in and with and to the cell a of for the of treatment to with and in and and the to the in to the with The on a for and in a to the with with of progranulin B. Yu G. J. et progranulin is a and is a of Biol. Chem. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar) by a with A with the and the and in the on to and with blocking the with to with the The with progranulin used to the of ASOs on cell in a of The with the and in of in for the and of to the The a with and the to with A FRET-based to the miR-29b with the and a GRN 3′ UTR with the 3′ in in with miR-29b and GRN 3′ UTR in of ASOs to the in the a with and and with and The of for and with GRN mice B. G. J. W. et mice as a novel for the of 2021; PubMed Scopus Google Scholar) on the and by as B. G. J. W. et mice as a novel for the of 2021; PubMed Scopus Google Scholar) by in a with a and and used for studies to of ASO ASO by the of mice with as and of protein improves and and in 2014; PubMed Scopus Google Scholar). mice and brain for protein and progranulin levels in of protein well a and as as K. P. F. et increase progranulin levels in the Grn mouse model of frontotemporal Scopus Google Scholar). in and a and with a that the ASO Miller A.J. et neuronal loss and reverses and in mice with Transl. 2017; PubMed Scopus Google Scholar), and and on with a approved by the and and are as as as in the with the in the The used in study are in the Gene the other the of study are the and its This K. P. and F. R. are of and for on GRN ASOs to treat frontotemporal The that no other of with the of We for for the for for and and of the for G. K. P. F. and A. D. N. G. S. S. A. Y. J. M. D. M. M. L. M. I. M. S. T. L. K. P. S. A. and A. D. N. G. I. M. S. T. L. B. R. K. P. F. J. E. S. A. P. A. C. F. and A. D. N. T. L. B. R. K. P. F. J. E. S. A. and A. D. N. A. D. N. S. A. P. A. C. F. and A. D. N. P. A. C. F. and A. D. N. This by from the of and to A. D. of for to A. D. to FTD to A. D. and of to P. A. to C. F. to and to C. F. to C. F. and to P. A. D. The is the of the and the of the of