Gene therapy for retinitis pigmentosa

Transcription

1 Gene therapy for retinitis pigmentosa Robert E MacLaren Professor of Ophthalmology University of Oxford & Honorary Consultant Vitreoretinal Surgeon Oxford University Hospitals, Moorfields Eye Hospital & Manchester Royal Eye Hospital REM is the Scientific Founder of Nightstar Therapeutics Inc. REM is a Consultant to Nightstar, Spark and Gyroscope Therapeutics. REM is listed as inventor on several gene therapy patents licenced for commercial use.

2 X-linked retinitis pigmentosa caused by RPGR 23 year old man, RPGR c.779-5t>g Outer nuclear layer (ONL) = photoreceptors External limiting membrane Hood et al. The transition zone between healthy and diseased retina in patients with retinitis pigmentosa. 2011

3 ATGAGGGAGCCGGAAGAGCTGATGCCCGATTCGGGTGCTGTGTTTACATTTGGGAAAAGTAAATTTGCTGAAAATAATCCCGGTAAATTCTGGTTTAAAAATGATGTCCCTG TACATCTTTCATGTGGAGATGAACATTCTGCTGTTGTTACCGGAAATAATAAACTTTACATGTTTGGCAGTAACAACTGGGGTCAGTTAGGATTAGGATCAAAGTCAGCCAT CAGCAAGCCAACATGTGTCAAAGCTCTAAAACCTGAAAAAGTGAAATTAGCTGCCTGTGGAAGGAACCACACCCTGGTGTCAACAGAAGGAGGCAATGTATATGCAACTGGT GGAAATAATGAAGGACAGTTGGGGCTTGGTGACACCGAAGAAAGAAACACTTTTCATGTAATTAGCTTTTTTACATCCGAGCATAAGATTAAGCAGCTGTCTGCTGGATCTA ATACTTCAGCTGCCCTAACTGAGGATGGAAGACTTTTTATGTGGGGTGACAATTCCGAAGGGCAAATTGGTTTAAAAAATGTAAGTAATGTCTGTGTCCCTCAGCAAGTGAC CATTGGGAAACCTGTCTCCTGGATCTCTTGTGGATATTACCATTCAGCTTTTGTAACAACAGATGGTGAGCTATATGTGTTTGGAGAACCTGAGAATGGGAAGTTAGGTCTT CCCAATCAGCTCCTGGGCAATCACAGAACACCCCAGCTGGTGTCTGAAATTCCGGAGAAGGTGATCCAAGTAGCCTGTGGTGGAGAGCATACTGTGGTTCTCACGGAGAATG CTGTGTATACCTTTGGGCTGGGACAATTTGGTCAGCTGGGTCTTGGCACTTTTCTTTTTGAAACTTCAGAACCCAAAGTCATTGAGAATATTAGGGATCAAACAATAAGTTA TATTTCTTGTGGAGAAAATCACACAGCTTTGATAACAGATATCGGCCTTATGTATACTTTTGGAGATGGTCGCCACGGAAAATTAGGACTTGGACTGGAGAATTTTACCAAT CACTTCATTCCTACTTTGTGCTCTAATTTTTTGAGGTTTATAGTTAAATTGGTTGCTTGTGGTGGATGTCACATGGTAGTTTTTGCTGCTCCTCATCGTGGTGTGGCAAAAG AAATTGAATTCGATGAAATAAATGATACTTGCTTATCTGTGGCGACTTTTCTGCCGTATAGCAGTTTAACCTCAGGAAATGTACTGCAGAGGACTCTATCAGCACGTATGCG GCGAAGAGAGAGGGAGAGGTCTCCAGATTCTTTTTCAATGAGGAGAACACTACCTCCAATAGAAGGGACTCTTGGCCTTTCTGCTTGTTTTCTCCCCAATTCAGTCTTTCCA CGATGTTCTGAGAGAAACCTCCAAGAGAGTGTCTTATCTGAACAGGACCTCATGCAGCCAGAGGAACCAGATTATTTGCTAGATGAAATGACCAAAGAAGCAGAGATAGATA ATTCTTCAACTGTAGAAAGCCTTGGAGAAACTACTGATATCTTAAACATGACACACATCATGAGCCTGAATTCCAATGAAAAGTCATTAAAATTATCACCAGTTCAGAAACA AAAGAAACAACAAACAATTGGGGAACTGACGCAGGATACAGCTCTTACTGAAAACGATGATAGTGATGAATATGAAGAAATGTCAGAAATGAAAGAAGGGAAAGCATGTAAA CAACATGTGTCACAAGGGATTTTCATGACGCAGCCAGCTACGACTATCGAAGCATTTTCAGATGAGGAAGTAGAGATCCCAGAGGAGAAGGAAGGAGCAGAGGATTCAAAAG GAAATGGAATAGAGGAGCAAGAGGTAGAAGCAAATGAGGAAAATGTGAAGGTGCATGGAGGAAGAAAGGAGAAAACAGAGATCCTATCAGATGACCTTACAGACAAAGCAGA GGTGAGTGAAGGCAAGGCAAAATCAGTGGGAGAAGCAGAGGATGGGCCTGAAGGTAGAGGGGATGGAACCTGTGAGGAAGGTAGTTCAGGAGCAGAACACTGGCAAGATGAG GAGAGGGAGAAGGGGGAGAAAGACAAGGGTAGAGGAGAAATGGAGAGGCCAGGAGAGGGAGAGAAGGAACTAGCAGAGAAGGAAGAATGGAAGAAGAGGGATGGGGAAGAGC AGGAGCAAAAGGAGAGGGAGCAGGGCCATCAGAAGGAAAGAAACCAAGAGATGGAGGAGGGAGGGGAGGAGGAGCATGGAGAAGGAGAAGAAGAGGAGGGAGACAGAGAAGA GGAAGAAGAGAAGGAGGGAGAAGGGAAAGAGGAAGGAGAAGGGGAAGAAGTGGAGGGAGAACGTGAAAAGGAGGAAGGAGAGAGGAAAAAGGAGGAAAGAGCGGGGAAGGAG GAGAAAGGAGAGGAAGAAGGAGACCAAGGAGAGGGGGAAGAGGAGGAAACAGAGGGGAGAGGGGAGGAAAAAGAGGAGGGAGGGGAAGTAGAGGGAGGGGAAGTAGAGGAGG GGAAAGGAGAGAGGGAAGAGGAAGAGGAGGAGGGTGAGGGGGAAGAGGAGGAAGGGGAGGGGGAAGAGGAGGAAGGGGAGGGGGAAGAGGAGGAAGGAGAAGGGAAAGGGGA GGAAGAAGGGGAAGAAGGAGAAGGGGAGGAAGAAGGGGAGGAAGGAGAAGGGGAGGGGGAAGAGGAGGAAGGAGAAGGGGAGGGAGAAGAGGAAGGAGAAGGGGAGGGAGAA GAGGAGGAAGGAGAAGGGGAGGGAGAAGAGGAAGGAGAAGGGGAGGGAGAAGAGGAGGAAGGAGAAGGGAAAGGGGAGGAGGAAGGAGAGGAAGGAGAAGGGGAGGGGGAAG AGGAGGAAGGAGAAGGGGAAGGGGAGGATGGAGAAGGGGAGGGGGAAGAGGAGGAAGGAGAATGGGAGGGGGAAGAGGAGGAAGGAGAAGGGGAGGGGGAAGAGGAAGGAGA AGGGGAAGGGGAGGAAGGAGAAGGGGAGGGGGAAGAGGAGGAAGGAGAAGGGGAGGGGGAAGAGGAGGAAGGGGAAGAAGAAGGGGAGGAAGAAGGAGAGGGAGAGGAAGAA GGGGAGGGAGAAGGGGAGGAAGAAGAGGAAGGGGAAGTGGAAGGGGAGGTGGAAGGGGAGGAAGGAGAGGGGGAAGGAGAGGAAGAGGAAGGAGAGGAGGAAGGAGAAGAAA GGGAAAAGGAGGGGGAAGGAGAAGAAAACAGGAGGAACAGAGAAGAGGAGGAGGAAGAAGAGGGGAAGTATCAGGAGACAGGCGAAGAAGAGAATGAAAGGCAGGATGGAGA GGAGTACAAAAAAGTGAGCAAAATAAAAGGATCTGTGAAATATGGCAAACATAAAACATATCAAAAAAAGTCAGTTACTAACACACAGGGAAATGGGAAAGAGCAGAGGTCC AAAATGCCAGTCCAGTCAAAACGACTTTTAAAAAACGGGCCATCAGGTTCCAAAAAGTTCTGGAATAATGTATTACCACATTACTTGGAATTGAAGTAA

4 RPGR is alternatively spliced in photoreceptors 5 Exons GGGAGGAGAG 16 Exons RPGR primary RNA transcript Intron 15 donor acceptor GUGAGU U1/6 srna NUCLEUS nuclear membrane AG nuclear membrane CYTOPLASM Ubiquitous RPGR mrna

5 RPGR is alternatively spliced in photoreceptors Intron 15 donor acceptor 5 Exons GGGAGGAGAG 16 Exons RPGR AG primary RNA transcript GUGAGU AG U1/6 srna nuclear membrane NUCLEUS nuclear membrane CYTOPLASM GUGAGU Photoreceptor specific RPGR mrna - RPGR ORF Exons Exons Ubiquitous RPGR mrna

6 RPGR gene therapy: problem 5 Exons GGCAG GA 3 AAV8.RPGR mrna GUGAGU AG U1/6 srna nuclear membrane NUCLEUS nuclear membrane CYTOPLASM Truncated RPGR mrna potentially toxic Wu et al. A long-term efficacy study of gene replacement therapy for RPGR-associated retinal degeneration. Hum Molecular Genetics 2016

7 RPGR gene therapy: solution 5 Exons GGCAG GA 3 AAV8.RPGR mrna U1/6 srna nuclear membrane GUGUCC Codons AGU=Serine UCC=Serine NUCLEUS nuclear membrane CYTOPLASM Correct full-length RPGR ORF15 mrna from AAV8 vector

8 Codon-optimised RPGR yields correct protein Western blot RPGR bands corresponding to the full length protein Dominik Fischer in Oxford Fischer et al., (2017) Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa. Molecular Therapy

9 ORF15 region is functional 5 Exons GGCAG GA 3 GAA = Glu GAG = Glu GGA = Gly GGG = Gly NH 2 Glu Gly Glu Gly Glu Gly COOH RPGR ORF15 translated into protein

10 RPGR glutamylation with TTLL5 Glu Gly Glu Gly Glu Gly TTLL5 RPGR ORF15 post-translational modification of protein

11 Glutamylation moves RPGR inner segment outer segment tubulin in photoreceptor cilium Glu Gly Glu Gly Glu Gly RPGR ORF15 post-translational glutamylation

12 Negative effects of ORF15 deletions Sun et al. Loss of RPGR glutamylation underlies pathology of TTLL5 mutations. Proc Natl Acad Sci 2016.

13 Codon optimised RPGR in vitro Dr Cristina Martinez Nuffield Laboratory of Ophthalmology, University of Oxford Full length RPGR protein with no splice variants in codon optimised RPGR construct (white arrow) CORRECT SPLICING Full length and fully glutamylated ORF15 seen with GT335 immunostaining in codon optimised RPGR CORRECT GLUTAMYLATION

14 RPGR gene therapy - XIRIUS A Phase 1/2 Retinal Gene Therapy for X-linked Retinitis Pigmentosa Using an Adeno-Associated Viral Vector (AAV8) Encoding RPGR AAV8.RK.coRPGR gene therapy Phase I/II clinical trial (first for XLRP) Multicentre UK (Oxford, Manchester, Southampton) and USA (Bascom Palmer, Miami) 6 cohort doses from 1 x 10 9 to 5 x genome particles in 100 µl (n=18 total) Primary outcome: safety and tolerability; Secondary outcome: efficacy compared with untreated eyes

15 First human RPGR gene therapy Oxford Eye Hospital, Oxford, Thursday 16 March 2017

16 First human RPGR gene therapy

17 Subjective visual field improvement dB Threshold Change in Mean Sensitivity (db) at Month Treated Untreated Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort Advanced degeneration Mild subretinal inflammation

18 Improved microperimetry by 1 month in cohort 3 Baseline Month 1 Month 3 Month 6 P1 P2 P3

19 Treatment effect is sustained Cohort 3 patients (n=3): Total number of Loci with Increase in Retinal Sensitivity Month Month Month >=1 db >=2 db >=3 db >=4 db >=5 db >=6 db >=7 db 0 >=1 db >=2 db >=3 db >=4 db >=5 db >=6 db >=7 db 0 >=1 db >=2 db >=3 db >=4 db >=5 db >=6 db >=7 db Increase in Retinal Sensitivity (db) from baseline treated eyes fellow eyes

20 Response in cohort 4 patient BASELINE 1 MONTH 0.5 db Treated 0.7 db Untreated 3.4 db Treated 0.5 db 3 MONTH 6 MONTH Untreated 6.6 db Treated 0.5 db Untreated 6.9 db Treated 0.5 db Untreated

21 A patient describes his experience This patient has given his written consent for this interview to be shown at Euretina

22 XIRIUS Trial for XLRP - Conclusions All 18 patients in six cohorts have been treated in the escalation phase of the XIRIUS trial Early proof of concept with durable dose related improvements seen as early as month 1 Proof of concept provides us with basis to move into the expansion phase this year Mild steroid-responsive sub-retinal inflammation has been observed in cohorts 4-5 (no vitritis) Sustained efficacy signal seen in 3/3 patients in cohort 3 and 2/6 patients in cohorts 4 and 5

23 Oxford Research Team & Funding