sirna therapy for TTR-related ocular amyloidosis

Transcription

1 sirna therapy for TTR-related ocular amyloidosis Masayoshi Tasaki 1, Hirofumi Jono 1, Mitsuharu Ueda 1, Ryuhei Hara 2, Konen Obayashi 1, Takahiro Kawaji 2, Alvarez Rene 3, Yoshimasa Mori 4, Taro Yamashita 5 and Yukio Ando 1 1 Department of Diagnostic Medicine, 2 Department of Ophthalmology and Visual Science and 5 Department of Neurology, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan. 3 Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA 4 Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.

2 Transthyretin (TTR)-related Familial amyloidotic polyneuropathy (FAP) FAP is characterized by amyloid deposition caused by amyloidogenic TTR (ATTR). Although ATTR is mainly produced by liver, ATTR is also produced by retinal pigment epithelium (RPE), choroid plexus, and pancreas. Ocular manifestations are commonly found and cause blindness. Abnormal conjunctival vessels Pupillary abnormalities Vitreous opacity Glaucoma

3 Ocular manifestations after liver transplantation 100% 80% 60% 40% 20% Amyloid deposition on the fringed pupil 100% 80% 60% 40% 20% Morphological change in the iris Liver transplantation: LT 0% Before LT 0% 1 y 3 y 5 y 7 y 10 y (after LT) Before LT 1 y 3 y 5 y 7 y 10 y (after LT) 100% Vitreous opacity 100% Glaucoma 80% 80% 60% 60% 40% 40% 20% 20% 0% Before LT 1 y 3 y 5 y 7 y 10 y (after LT) 0% Before LT 1 y 3 y 5 y 7 y 10 y (after LT) Unpublished Data Ocular New manifestations therapeutic become strategies advanced to prevent after the progression liver transplantation, of because ATTR ocular is produced manifestations by the RPE are cells. urgently needed. Ando E et al. Ann Intern Med 2001; 135: 931-2

4 Retinal laser photocoagulation for ocular manifestations Retinal laser photocoagulation is a common treatment used for diabetic retinopathy. This method could suppress ATTR production by destroying the RPE Retinal laser photocoagulation (-) 1 year 2 years 3 years Retinal laser photocoagulation (+) 1 year 2 years Kawaji T et al. Ophthalmology 2010 Laser photocoagulation, which reduced the RPE, prevented the progression of amyloid deposition in the vitreous Effective suppression of ATTR expression in the RPE may become a novel therapy for ocular amyloidosis 3 years

5 Small interfering RNA (sirna) therapy for ocular amyloidosis TTR-specific sirna TTR sirna ATTR production in the RPE cells RPE TTR sirna ATTR Ocular manifestations production Amyloid deposition

6 Purpose To evalutate the potential utilization of sirna treatment as a novel therapy for ocular amyloidosis

7 Ratio of ATTR V30 M mrna copies to rat GAPDH mrna copies ( 10-3 ) Materials and methods sirna (Alnylam) : Human TTR, rat TTR- and control sirna in vitro: Human RPE cell line (ARPE-19) in vivo: Dark Agouti (DA) rats ATTR V30M transgenic (TG) rats Evaluation of TTR expression: real-time quantitative PCR 100,000 Human ATTR V30M expression in various tissues Rat albumin enhancer/ promoter Human ATTR V30M 10,000 1, Liver Brain Eye Lung Kidney

8 n human TTR/ human GAPDH relative expression human TTR/ human GAPDH mrna relative expression Effect of human TTR sirna on human TTR expression < ARPE-19 > Control sirna Human TTR Human TTR sirna Rat TTR sirna A (1 nm ) A (1 nm ) 1 nm 10 nm 50 nm A (1 nm ) (10 nm ) (10 nm ) (10 nm ) (50 nm ) (50 nm ) (50 nm )

9 rat TTR/ rat G APDH relative expression Rat TTR/ rat GAPDH mrna relative expression Effect of rat TTR sirna on endogenous rat TTR expression 2.0 < RPE from normal rats > Endogenous rat TTR P< No treat Saline n.t saline controlsir N A TTR sir N A d Control sirna Rat TTR sirna

10 relative expression Human TTR/ rat GAPDH mrna relative expression Effect of human TTR sirna on human ATTR expression < RPE from ATTR V30M TG rats > 2.0 Human ATTR V30M No Treat Saline Control sirna TTR sirna n.t saline controlsir N A TTR sir N A

11 <In vitro> Summary Human TTR sirna specifically reduced human TTR mrna in the RPE cell line No significant inflammatory responses were observed in the sirna-transfected cells <In vivo> Rat TTR sirna significantly reduced endogenous rat TTR mrna expression in the RPE Expression of ATTR mrna was efficiently and specifically reduced by human TTR sirna in the RPE

12 Future plans Optimize sirna delivery - determine dose- and time- dependent effect - Utilize transfection reagents Improve injection methodology - subconjunctival space, subretina, etc. - increase volume of inoculum Monitor for potential side effects - Evaluation of inflammation, off-target effects, toxicity, etc. TTR sirna progression A new therapy for TTR-related ocular amyloidosis

13 Conclusion sirna treatment may become a novel therapy for TTR-related ocular amyloidosis.