RNA-Seq guided gene therapy for vision loss. Michael H. Farkas

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1 RNA-Seq guided gene therapy for vision loss Michael H. Farkas

2 The retina is a complex tissue Many cell types Neural retina vs. RPE Each highly dependent on the other Graw, Nature Reviews Genetics,

3 Inherited Retinal Degenerations 212+ disease genes identified to date Genetically and clinically diverse (Modified from Berger, et al 2010)

4 Retinitis Pigmentosa (RP) RP is the most common form of inherited blindness Affects 1: individuals worldwide Patients typically present in early adulthood with progressive night blindness and loss of peripheral vision Complete vision loss occurs later in life Currently, nearly 50 genes implicated in non-syndromic RP Many expressed specifically in the retina

5 mrna Splicing and RP 6 spliceosome-associated genes identified as important causes of dominant RP: -PRPF3, PRPF6, PRPF8, PRPF31, RP9, SNRNP200 All function in the U4/U6/U5 tri-snrnp

6 RNA Splicing Factor RP How do mutations in spliceosome components cause retina-specific disease? Hypotheses: 1. via production of specific altered transcripts that are pathogenic in the retina 2. via global alterations in splicing that affect the retina most Goals: Test these hypotheses in gene targeted Prpf3, Prpf8 and Prpf31 mice, and human RPE cells Develop therapeutic strategies for these common forms of RP

7 RPE and Retinal Degeneration in Prpf Mutant Mice (Graziotto, et al 2011)

8 Defective Function of Prpf Mutant RPE (Farkas, et al AJP, 2014)

9 RNA-Seq Library Preparation RPE is a single cell layer Tissue/RNA is limited in mice Have nanogram levels of total RNA to start Traditional protocols use microgram levels of total RNA Protocol is optimized for 200 ng of total RNA, but can be adjusted to use less

10 RNA-Seq Unified Mapper (RUM) Alignment of RNA-Seq reads was problematic when RNA-Seq was first developed Requires accurately mapping reads over splice junctions that can span hundreds of kilobases Many algorithms have since been developed RUM is one of the most accurate in mapping reads Provides data for exons, introns, splice junctions, and transcripts Very well suited for detecting novel splicing variants novel isoforms and novel genes Grant, et al., Bioinformatics, 2011

11 Aberrant Splicing - RPE How many aberrant transcripts are detected in the mutant RPE transcriptomes? Prpf3 179 Prpf8 44 Prpf31 24 No aberrant transcripts are shared amongst the models, further suggesting that disease pathogenesis is caused by different mechanisms aberrant transcripts are detected in the retina, brain, and muscle samples, suggesting aberrant splicing is widespread.

12 Altered Splicing - Rgr

13 Altered Splicing - Rgr

14 # of Novel Features Characterization of the Human and Mouse Retinal Transcriptome RNA-Seq libraries prepared from human and mouse retina, and mouse RPE and ARPE-19 cells. Approximately 80% of all annotated exons are expressed in the retina. An additional 3.5 Mb of novel features were found to be expressed. Large number of novel splicing events detected by RUM: Human retina 79,915 Mouse retina 47,078 Mouse RPE 34,061 ARPE-19 cells 32, Farkas, et al., BMC Genomics, Novel Exons Exon Skipping Alternate 3'/5' Splice Site Reads (Millions)

15 % of Total Junctions Novel Exons in the Retina Novel > 10x Novel 5-10x Novel 2-5x Novel = Annotated Annotated 2-5x Annotated 5-10x Annotated > 10x Nearly 30,000 novel exons identified in the human retina. ~5% of the novel exons compose the major isoform. 14% are predicted to maintain an open reading frame (ORF). Farkas, et al., BMC Genomics, 2013 Ratio of Novel to Annotated Junctions

16 Novel Exon Skipping Exon skipping is the most prevalent alternative splicing event. 82% of the novel exon skipping events skip one exon 17% are predicted to maintain the ORF

17 Novel Alternate Splice Sites Nearly 19,000 novel alternate splice sites identified. ~25% are predicted to maintain an open reading frame (ORF). Conclusion current transcriptome annotations are under-represented and comprehensive annotation can be important for finding mutations Novel exons have been found to harbor mutations in IRD genes (ABCA4)

18 What do transcriptome analyses tell us? Large number of novel splicing events in neural retinal transcriptomes Human retina 79,915 19,637 novel internal exons Including 206 in 99 of the known IRD disease genes Data are available via OGI website Mouse retina 47,078 Large number of novel splicing events in the RPE Mouse RPE 34,061 Human ARPE19 cells 32,178

19 15,000 novel events were chosen from RNA-Seq data Exon skipping, novel exons, alternate 3 /5 splice sites. Read depth as low as 1 Baits developed using Agilent s SureSelect Targeted RNA Capture System. Sequenced on a HiSeq2000

20 Large Scale Validation of Novel Transcriptome Features Evaluated 15,000 novel splicing events by targeted RNA capture 99% of the novel events were validated in retinal samples 71%, 61%, and 58% events detected in brain, liver, and muscle Nearly 2,000 novel splicing events may be restricted to the retina Data are available via OGI website (Farkas et al BMC Genomics 2013)

21 PRPF31-associated RP Mutations in PRPF31 are the second most common cause of adrp 50, ,000 affected people worldwide Pathogenesis of disease due to haploinsufficiency Mutations can be nonpenetrant (Rivolta et al, Human Mutation, 2006)

22 Functional validation of PRPF31 variants Family with adrp underwent whole exome sequencing. PRPF31 c.-9+1g>c variant identified as top candidate for potential pathogenicity. First base of donor site in intron 1.

23 Relative Expression Functional validation of PRPF31 variant X X * * 0.0 Control *p < 0.05

24 Functional validation of PRPF31 variant A* B C D

25 Functional validation of PRPF31 variants A* B C D A* B No splice variants produced premature stop codons. Additional studies, including knock-in and patient ips cells, are in progress to study mrna stability and affect on phagocytosis. C D

26 CRISPR/Cas9 Knockout of PRPF31 Guide RNAs (grna) designed to exon 6 of PRPF31 ips cells and ARPE-19 co-transfected with grna and Cas9:GFP GFP positive cells were single cell sorted into a 96-well plate Sanger validated: 25% heterozygous sites undergoing non-homologous end joining, which included 1-13 bp deletions No homozygous knockout clones observed consistent with haploinsufficiency mechanism

27 Relative PRPF31 Expression Decreased PRPF31 Expression Levels qpcr primers designed to 3 locations in PRPF31 γ-tubulin 1 (TUBG1) used as a control due to its similar expression level in ARPE-19 cells 5 lines chosen for analysis ** ** ** *P < 0.05 **P < 0.01 * * 0.0 Control GE31-1 GE31-2 GE31-3 GE31-6 GE31-7

28 Relative Phagocytosis Functional Characterization - Phagocytosis Each line plated in triplicate wells of a 48 well plate. Assayed 3 weeks after cells reached confluence to ensure polarization. FITC-labeled photoreceptor outer segments phagocytosed by ARPE-19 were counted using flow cytometry Control GE31-1 GE31-2 GE31-3 GE31-6 GE31-7

29 Relative POS Uptake Functional Characterization - Phagocytosis Control GE31-1 GE31-2 GE31-3 GE31-6 GE Hour 2 Hour 3 Hour Incubation Time While POS uptake begins to reach a maximum after 3 hours, the knockout lines seem to be increasing in the rate of uptake. Is phagocytosis slow, rather than non-existent in PRPF31 models?

30 Relative POS Uptake AAV-PRPF31 Gene Augmentation * No AAV-PRPF31 MOI = 10,000 MOI = 15, WT GE31

31 Acknowledgements Massachusetts Eye and Ear Pierce Lab Dr. Libby Au Dr. Kinga Bujakowska Dr. Donna Garland Dr. Chari Fernandez Godino Matt Maher Daniel Navarro Dr. Eric Pierce Emily Place Maria Sousa Dr. Scott Greenwald Dr. Joe White Dr. Qi Zhang Liu Lab Mihoko Leon Dr. Qin Liu Comander Lab Dr. Jason Comander Ally Lansdorf Harvard Stem Cell Institute Cowan Lab Dr. Chad Cowan Dr. Derek Peters Jen Shay Johns Hopkins University Zack Lab Dr. Melissa Liu Dr. Tomo Masuda Dr. Don Zack Qian Lab Dr. Jiang Qian Dr. Jun Wan University of Wisconsin-Madison Gamm Lab Dr. Beth Capowski Dr. David Gamm Anna Petelinsek Jishnu Saha Institut de la Vision Dr. Shomi Bhattacharya Dr. Emeline Nandrot UCLA Dr. Xiaowu Gai University of Pennsylvania Dr. Greg Grant Funding NEI NRSA Foundation Fighting Blindness MEEI

32 Agilent products are for Research Use Only. Not for use in diagnostic procedures.