- Email: [email protected]
CAS9 TO MODEL ALZHEIMER’S DISEASE
P1142
Poster Presentations: Wednesday, July 27, 2016
network and visual network with AD group showing the largest values in these networks (Fig. 2). Within the homotopic connectivity, long and short intrahemispheric and heterotopic connectivity, there were significant FCFS differences between groups (Fig. 3(A, B)). Finally, we observed significant negative correlations between the FCFS values and MMSE in default mode network and long intrahemispheric connectivity group, and between the FCFS values and MoCA in the long intrahemispheric connectivity group (P < 0.05, corrected) (Fig. 4). Conclusions: The disruption of brain dynamic functional connectivity networks in AD continuum could be revealed by resting-state fNIRS. These findings highlight the potential of FCFS of resting-state fNIRS as the sensitive biomarker for AD.
We show that HDR accuracy is dramatically increased by incorporating silent CRISPR/Cas-blocking mutations together with pathogenic mutations, and establish a method we termed “CORRECT” for scarless editing. Furthermore, by characterizing and exploiting a stereotyped inverse relationship between the incorporation rate of a mutation and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction require targeting a guide RNA close to the intended mutation, whereas heterozygous introduction can be achieved by distance-dependent suboptimal mutation incorporation or by using mixed repair templates. Using this approach, we establish human induced pluripotent stem cells (iPSCs) with homozygous and heterozygous dominant EOAD mutations in amyloid precursor protein (APPSwe) and presenilin 1 (PSEN1M146V) and derive cortical neurons, which display genotype-dependent disease-associated phenotypes. Conclusions: Our findings enable efficient introduction of homo- and heterozygous disease-associated mutations with CRISPR/Cas9, facilitating the study of molecular mechanisms of dementia in human in vitro models. P4-285
ß-AMYLOID-OLIGOMER-INDUCED ALZHEIMER’S MODEL SHOWS SYNAPTIC DEGRADATION FOR USE IN TARGET VALIDATION AND DRUG DEVELOPMENT
Pascal Goetghebeur, SynAging SA, Vandoeuvre-les-Nancy, France. Contact e-mail: [email protected] Background: Oligomeric forms of b-amyloid peptide (AbO) are P4-284
EFFICIENT INTRODUCTION OF DISEASERELATED MUTATIONS INTO HUMAN IPSCS USING CRISPR/CAS9 TO MODEL ALZHEIMER’S DISEASE
Dylan Kwart1, Dominik Paquet1, Antonia Chen1, Andrew Sproul2, Samson Jacob3, Shaun Teo1, Kimberly Moore Olsen1, Andrew Gregg1, Scott Noggle3, Marc Tessier-Lavigne1, 1The Rockefeller University, New York, NY, USA; 2Columbia University, New York, NY, USA; 3The New York Stem Cell Foundation, New York, NY, USA. Contact e-mail: dpaquet@ rockefeller.edu Background: CRISPR/Cas9 allows sequence-specific gene editing
in many organisms and is a promising tool to generate models of human diseases, e.g. in human pluripotent stem cells (iPSCs). CRISPR/Cas9 introduces double stranded breaks (DSBs) with high efficiency and locus specificity, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific indel mutations, useful for generating gene knockouts. In rare cases, DSBs can also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knock-outs through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modeling genetic disorders by introducing disease-associated mutations. Furthermore, targeted mutation knock-in at single alleles, to model diseases caused by heterozygous mutations, such as early-onset Alzheimer’s disease (EOAD), has not been reported. Methods: We developed an efficient CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy into iPSCs. Results:
widely accepted as the initial cause for neurodegeneration in Alzheimer’s disease (AD), related mild-cognitive impairment (MCI) and dementia, also triggering neuro-inflammatory processes and other complications. To reduce the significant attrition during drug discovery for neurodegenerative diseases, translational in vitro and in vivo models are essential. Methods: Here, we report highly reproducible in vitro and in vivo AD models, based on the neurodegenerative effects of a single injection of AbO preparations. Results: Our AbO preparations induce dramatic neuronal cell death (monitored using multiple read-outs) in rodent primary neurons from different brain areas, as well as in human neurons derived from iPS cells. AbO-induced neuronal apoptosis is associated with neuro-inflammatory processes, increased production of pro-inflammatory cytokines, and impairment of sphingolipid cell signaling. In rodent models (mice and rats), a single brain injection of minute amounts of AbO results in dramatic and fast impairment of cognitive functions, associated with synaptic dysfunctions. A remarkable loss of synaptic marker proteins has indeed been found. Conclusions: Unlike transgenic models, our acute models imitate sporadic AD, are easily scalable to statistically meaningful animal numbers, and are successfully used for fast and cost effective phenotypic validation of preclinical candidates.
P4-286
RETROGRADE AXONAL DEGENERATION CAUSED BY AMYLOID BETA INJECTION IN AXONAL TERMINALS OF P301L MICE
Shu-Wei Sun1, Christopher Nishioka2, Hsiao-Fang Liang1, 1Loma Linda University, Loma Linda, CA, USA; 2University of California in Riverside, Riverside, CA, USA. Contact e-mail: [email protected]
Poster Presentations: Wednesday, July 27, 2016
network and visual network with AD group showing the largest values in these networks (Fig. 2). Within the homotopic connectivity, long and short intrahemispheric and heterotopic connectivity, there were significant FCFS differences between groups (Fig. 3(A, B)). Finally, we observed significant negative correlations between the FCFS values and MMSE in default mode network and long intrahemispheric connectivity group, and between the FCFS values and MoCA in the long intrahemispheric connectivity group (P < 0.05, corrected) (Fig. 4). Conclusions: The disruption of brain dynamic functional connectivity networks in AD continuum could be revealed by resting-state fNIRS. These findings highlight the potential of FCFS of resting-state fNIRS as the sensitive biomarker for AD.
We show that HDR accuracy is dramatically increased by incorporating silent CRISPR/Cas-blocking mutations together with pathogenic mutations, and establish a method we termed “CORRECT” for scarless editing. Furthermore, by characterizing and exploiting a stereotyped inverse relationship between the incorporation rate of a mutation and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction require targeting a guide RNA close to the intended mutation, whereas heterozygous introduction can be achieved by distance-dependent suboptimal mutation incorporation or by using mixed repair templates. Using this approach, we establish human induced pluripotent stem cells (iPSCs) with homozygous and heterozygous dominant EOAD mutations in amyloid precursor protein (APPSwe) and presenilin 1 (PSEN1M146V) and derive cortical neurons, which display genotype-dependent disease-associated phenotypes. Conclusions: Our findings enable efficient introduction of homo- and heterozygous disease-associated mutations with CRISPR/Cas9, facilitating the study of molecular mechanisms of dementia in human in vitro models. P4-285
ß-AMYLOID-OLIGOMER-INDUCED ALZHEIMER’S MODEL SHOWS SYNAPTIC DEGRADATION FOR USE IN TARGET VALIDATION AND DRUG DEVELOPMENT
Pascal Goetghebeur, SynAging SA, Vandoeuvre-les-Nancy, France. Contact e-mail: [email protected] Background: Oligomeric forms of b-amyloid peptide (AbO) are P4-284
EFFICIENT INTRODUCTION OF DISEASERELATED MUTATIONS INTO HUMAN IPSCS USING CRISPR/CAS9 TO MODEL ALZHEIMER’S DISEASE
Dylan Kwart1, Dominik Paquet1, Antonia Chen1, Andrew Sproul2, Samson Jacob3, Shaun Teo1, Kimberly Moore Olsen1, Andrew Gregg1, Scott Noggle3, Marc Tessier-Lavigne1, 1The Rockefeller University, New York, NY, USA; 2Columbia University, New York, NY, USA; 3The New York Stem Cell Foundation, New York, NY, USA. Contact e-mail: dpaquet@ rockefeller.edu Background: CRISPR/Cas9 allows sequence-specific gene editing
in many organisms and is a promising tool to generate models of human diseases, e.g. in human pluripotent stem cells (iPSCs). CRISPR/Cas9 introduces double stranded breaks (DSBs) with high efficiency and locus specificity, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific indel mutations, useful for generating gene knockouts. In rare cases, DSBs can also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knock-outs through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modeling genetic disorders by introducing disease-associated mutations. Furthermore, targeted mutation knock-in at single alleles, to model diseases caused by heterozygous mutations, such as early-onset Alzheimer’s disease (EOAD), has not been reported. Methods: We developed an efficient CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy into iPSCs. Results:
widely accepted as the initial cause for neurodegeneration in Alzheimer’s disease (AD), related mild-cognitive impairment (MCI) and dementia, also triggering neuro-inflammatory processes and other complications. To reduce the significant attrition during drug discovery for neurodegenerative diseases, translational in vitro and in vivo models are essential. Methods: Here, we report highly reproducible in vitro and in vivo AD models, based on the neurodegenerative effects of a single injection of AbO preparations. Results: Our AbO preparations induce dramatic neuronal cell death (monitored using multiple read-outs) in rodent primary neurons from different brain areas, as well as in human neurons derived from iPS cells. AbO-induced neuronal apoptosis is associated with neuro-inflammatory processes, increased production of pro-inflammatory cytokines, and impairment of sphingolipid cell signaling. In rodent models (mice and rats), a single brain injection of minute amounts of AbO results in dramatic and fast impairment of cognitive functions, associated with synaptic dysfunctions. A remarkable loss of synaptic marker proteins has indeed been found. Conclusions: Unlike transgenic models, our acute models imitate sporadic AD, are easily scalable to statistically meaningful animal numbers, and are successfully used for fast and cost effective phenotypic validation of preclinical candidates.
P4-286
RETROGRADE AXONAL DEGENERATION CAUSED BY AMYLOID BETA INJECTION IN AXONAL TERMINALS OF P301L MICE
Shu-Wei Sun1, Christopher Nishioka2, Hsiao-Fang Liang1, 1Loma Linda University, Loma Linda, CA, USA; 2University of California in Riverside, Riverside, CA, USA. Contact e-mail: [email protected]