Targeted Epigenetic Modification Kyle Fink, PhD UC Davis Stem Cell Program Adjunct Assistant Professor, Department of Neurology

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1 TALE CRISPR/Cas9 Targeted Epigenetic Modification Kyle Fink, PhD UC Davis Stem Cell Program Adjunct Assistant Professor, Department of Neurology Derived from plant pathogenic bacteria from the genus Xanthomas One of many DNA-targeting proteins Each repeat comprises amino acids. Can be rapidly synthesized to target any base pair sequence Highly efficient and specific with minimal off-target effects Original Cas9 derived from Streptococcus pyogenes Uses a synthetic guide RNA (grna) to deliver the Cas9 to a desired location Cas9 can be used interchangeably with different grna allowing for rapid targeting and flexibility Both systems can be constructed with a variety of transcription factors to epigenetically regulate gene expression (i.e., nucleases, activators, repressors) Fink, KD 1

2 Transcription Activator-like Effector (Graphic Summary) A. GM2151 B. GM2167 C. GM D. GM4723-no SNP E. GM4737-no SNP F. GM4281b-biallelic SNP 1.1 Human HD fibroblasts confirmed to have SNP rs display significant allele-specific reduction of the mutant allele (red bar) without significantly altering healthy allele expression (blue bar). Fink, KD 2

3 Relative Allele Expression Relative Allele Expression A. B. C Primary Adult YAC128 Striatal Neuron Healthy Mutant # 1.4 Primary Adult YAC128 Cortical Neuron Healthy Mutant #. Wildtype Tg EV Tg TALE Wildtype Tg EV Tg TALE Primary cortical and striatal neurons cultured from a 1 month old YAC128 mouse treated with TALE. Fink, KD 3

4 Relative Allele Expression Relative Allele Expression A. B. Healthy Mutant TurboTALE Allele Expression C. TurboFect D. WT Tg Cortex + Cortex - Striatum + Striatum - IVV TALE Allele Expression 1.4 Healthy Mutant InVivofectamine WT Tg Cortex + Cortex - Striatum + Striatum - In vivo injection of the TALE plasmid and synthetic RNA using TurboFect and Invivofectamine. Fink, KD 4

5 Fluorescent Intensity Volume Fluorescent Intensity Volume in um3 A. D. Average Volume B. F2 E. F2 F1 F18 IVV Turbo Average Intensity F F2 F1 F18 IVV Turbo C. F IntensityVolume F18 F2 F1 F18 IVV Turbo Quantification of TALE biodistribution and expression following LNP encapsulation and unilateral injection. Fink, KD 5

6 Relative Allele Expression Relative Allele Expression MAB2166/Actin Striatal Co-Localization Cortical Co-Localization Cortical and Striatal and Co-localization A. B. C. NeuN TALE F2 F1 F18 D. E. F. Darpp32 TALE LNP TALE Striatal Allele Expression Tg F2 F1 F18 F2 F1 F18 G. H. I. LNP TALE Cortical Allele Expression Tg F2 F1 F18 1 LNP TALE Protein Quantification WT Tg F18 Co-localization of the TALE with cortical and striatal neurons with TaqMan SNP genotyping for allele expression and Western Blot for protein quantification. Fink, KD 6

7 Relative Gene Expression Relative Gene Expression Relative Gene Expression A Striatal P53 Injected Contralateral F2 F1 F18 Tg WT B. Striatal Bax F2 F1 F18 Tg WT C. 1.5 Striatal Wig1 Toxicity screen following injection of LNP encapsulated TALE..5 F2 F1 F18 Tg WT Fink, KD 7

8 CDKL5 mrna fold change (relative to GAPDH) Other Disease Indications CDKL5 deficiency TALE and dcas9 Angelman Syndrome MSC Delivery of S1 Anti-HA blot SHSY-5Y Adipose MSC transduced with Lentivirus S1-mCherry 4 days posttransduction - 6 kda band Treatment Gene Activation: Gene Silencing: Loss-of-function Angelman s Syndrome Zinc Finger approach CDKL5 deficiency (infantile epilepsy) TALE and CRISPR activation studies Gain-of-function diseases or disorders Huntington s disease TALE and CRISPR silencing studies Potential genes implicated in cancer Fink, KD 8

9 Thank you! Jan Nolta Director Stem Cell Program Vicki Wheelock Director Huntington s disease clinic David Segal Associated Director Genome Center Peter Deng Anvita Komarla Joey Aprile Megan Cheng Sharon Burk Thuy Nguyen Jasmine Carter Sakereh Carter Support for this project was provided by a NIH NRSA Postdoctoral Fellowship F32NS9722 (Fink) NIH Director s transformative award 1R1GM99688 (Nolta) NIH NIGMS Predoctoral Fellowship T32GM9968 (Deng) The Stewart s and Dake Family Gift (Nolta/Fink) Help4HD International LouLou Foundation, UPenn Orphan Disease Center (Nolta/Fink) Philanthropic donors from the HD community, including the Roberson family, WeHaveAFace.org and TeamKJ Fink, KD 9