QRX-704, a novel antisense oligonucleotide therapy ... · Institute of Science and Technology...
1
In vivo evaluation of QRX-704 activity Dose-response evaluation of QRX-704 activity in YAC128 HD model administered by intracerebroventricular injection, assessed by droplet digital PCR (Figure 6A). Biodistribution and astrocytosis was assessed in CD1 WT mice by fluorescent in situ hybridization (Figure 6B) and immunohistochemical staining for GFAP (Figure 6C). Generation of exon 12 isoform-specific monoclonal and polyclonal antibodies, directed against peptides for wt exon 12 or truncated Δ12 (Figure 7A), tested by western blot analysis (Figure 7B). QRX-704 was administered to FVB-YAC128 mice and sacrificed after 14 days for protein analysis, using crude antibody supernatant, which allowed detecting HTT Δ12 protein (Figure 8). 5 0 1 00 2 00 4 00 5 0 1 00 2 00 4 00 5 0 1 00 2 00 4 00 5 0 1 00 2 00 4 00 5 0 1 00 2 00 4 00 0 20 40 60 Droplet digital PCR Dose ( g) HTT 12 mRNA (% of total HTT) Scrambled QRX-704 Cortex Striatum Hippocampus Cerebellum Midbrain Figure 6A. FVB-YAC128, 2 weeks after single ICV administration Figure 6B. CD1 mouse 14 days after ICV-administration of QRX-704 (400 µg) QRX-704-FISH DAPI 1 mm 10 µm Figure 6C. GFAP immunohistochemistry aCSF 28 days 400 µg 7 days 100 µg 7 days 400 µg 14 days 200 µg 7 days 400 µg 28 days GFAP DAPI Figure 7B. COS7 cells overexpressing HTT WTex12/Δ12 isoforms anti-WTex12 #1187 (Rabbit poly) anti-total HTT MAB2166 anti-Δ12 #26E7 (Mouse mono) anti-total HTT D7F7 Figure 7A. Isoform specific antibodies Anti-wtEx12 Anti-Δ12 Results (continued) HTT Δ12 is resistant to HTT586 caspase-6 cleavage Caspase-6 cleavage of HTT Δ12 isoform assessed in lysates from COS7 cells expressing cDNA (Figure 3A), or HD fibroblasts transfected with QRX-704 (Figure 3B), followed by incubation with recombinant active caspase-6. HTT Δ12 maintains overall folding and biochemical characteristics of canonical HTT Biochemical analysis of purified 23Q or 78Q WT exon 12 or Δ12 HTT isoforms derived in Sf9 insect cells, assessed by SDS- and native PAGE (Figure 4A), CD spectroscopy (Figure 4B) and PTM analysis (Figure 4C). HTT WT exon 12 or HTT Δ12 overexpressed in SK-N-SH or COS7 cells indicates normal intracellular localization (Figure 5A), and unfolding/digestion pattern in pulse proteolysis (Figure 5B). Figure 3B. HD Fibroblasts (CAG40/50) Anti HTT MAB2166 (N-terminal) Figure 4C. Post translational modification mapped by mass spectrometry Phosphorylated sites with Mod Score >19 displayed Figure 4B. CD-spectroscopy Monomeric protein -40 -30 -20 -10 0 10 20 30 40 190 200 210 220 230 240 250 260 CD [mdeg] Wavelength [nm] HTT 23Q WTex12 HTT 23Q Δ12 HTT 78Q WTex12 HTT 78Q Δ12 Figure 4A. PAGE of purified proteins 5 % SDS-PAGE 5 % Native PAGE Figure 5A. Intracellular localization - SK-N-SH cells overexpressing HTT isoforms HTT WTex12 HTT Δ12 Anti-HTT Hoechst Figure 5B. Pulse proteolysis [Thermolysin] (mg/ml) Anti-HTT MAB2166 -2 -1 0 0.0 0.5 1.0 Western blot quantification Log [Thermolysin] (mg/ml) Full length HTT (a.u.) HTT WTex12 HTT ∆12 Introduction QRX-704 is a novel antisense oligonucleotide-based therapeutic approach, aiming to mitigate mutant Huntingtin (mHTT) toxicity, while maintaining physiological HTT function. Proteolytic cleavage of mHTT generates toxic N-terminal fragments that are hypothesized to be the main contributor to the pathogenesis of Huntington disease (HD). These fragments are formed from a cascade of proteolytic cleavages, initiated by caspase-6 cleavage at position D586. Importantly, HTT586 cleavage is required for HD-like phenotypes in the YAC128 mouse model 1 . Moreover, mHTT cleavage in turn increases caspase-6 activity, driving pathology in a toxic forward-feedback loop 2 (Figure 1A). While toxicity stemming from mHTT cleavage is the primary pathogenic mechanism, data indicates that HTT loss-of-function exacerbates pathology 3 . In addition, maintaining wild type HTT function may be critical for safety of HTT-targeted therapeutics when treating patients for a long period of time. Material and Methods QRX-704 splice-switching activity was tested and optimized in cultured HD fibroblasts (GM04857) and HD iPSC-derived mature neural cultures (ND42223), both from Coriell Institute, assessed by droplet digital PCR and western blotting. Biophysical and biochemical characterization of HTT Δ12 was performed using purified protein, with caspase-6 cleavage assays, CD-spectroscopy , and post-translational modification mapping. Intracerebroventricular (ICV) bolus administration of QRX-704 in in wild type and YAC128 mice, was used to study pharmacodynamic activity , quantified by droplet digital PCR, biodistribution assessed by fluorescent in situ hybridization, reactive gliosis was studied by immunohistochemistry. To better quantify HTT Δ12 protein, exon 12 isoform-specific antibodies were developed and used to detect HTT Δ12 in YAC128 brain tissue. Results QRX-704 sequence optimization in HD fibroblasts and iPSC-neurons Oligonucleotide lead candidate sequences screened and optimized in HD fibroblasts, assessed by droplet digital PCR and western blot (Figure 2A). Results were verified in HD iPSC neural culture (Figure 2B). Figure 1A. Caspase-6 cleavage of mHTT drives generation of toxic small N-terminal fragments in forward feedback loop Figure 1B. QRX-704 activates alternative exon 12 splice isoform, removing HTT586 caspase-6 site Objectives QRX-704 functions at the pre-mRNA level by activating an alternative HTT splice-isoform (HTT Δ12), removing the critical HTT586 caspase-6 cleavage site, thus preventing formation of toxic N-terminal fragments 4 (Figure 1B). The aim of the study is to pharmacologically characterize QRX-704 for preclinical development, and describe the novel HTT Δ12 isoform. Figure 2A. RNA modulation in HD fibroblasts Ladder Non transfected QRX-704 0 8 16 24 32 0 20 40 60 Droplet digital dPCR [QRX-704 transfected] (nM) HTT ∆12 mRNA (% of total HTT) QRX-704A QRX-704B QRX-704C Δ12 WT exon 12 Δ12 WT exon 12 Non- transfected QRX-704A (nM) 8 16 32 QRX-704B (nM) 8 16 32 QRX-704C (nM) 8 16 32 Huntingtin (MAB2166) Figure 2B. RNA modulation in HD iPSC neurons QRX-704, a novel antisense oligonucleotide therapy designed to prevent HD pathology while maintaining HTT function Pontus Klein 1 , Zhana Karneva 1 , Lodewijk Toonen 2 , Hyeongju Kim 3 , Nicholas Caron 4 , Linda van der Graaf 2 , Levi Buil 1 , Geert van der Horst 1 , Herma Anthonijsz 1 , Frits van der Ham 1 , Wouter Beumer 1 , Michael Hayden 4 , Ji-Joon Song 3 , Willeke van Roon-Mom 2 , Gerard Platenburg 1 (1) ProQR Therapeutics, Zernikedreef 9, 2333 CK Leiden, The Netherlands, (2) Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2300RC Leiden, The Netherlands, (3) Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea, (4) Centre for Molecular Medicine and Therapeutics (CMMT), CFRI, Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4 Canada Literature • Graham, Rona K., et al. “Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin.” Cell 125.6 (2006): 1179-1191 • Graham, Rona K., et al. “Cleavage at the 586 amino acid caspase-6 site in mutant huntingtin influences caspase-6 activation in vivo.” Journal of Neuroscience 30.45 (2010): 15019-15029. • Bečanović, Kristina, et al. “A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic modifier of Huntington disease.” Nature neuroscience 18.6 (2015): 807. • Evers, Melvin M., et al. “Preventing formation of toxic N-terminal huntingtin fragments through antisense oligonucleotide-mediated protein modification.” Nucleic acid therapeutics 24.1 (2014): 4-12. SEPTEMBER 2018 Conclusion QRX-704 constitutes a novel therapeutic approach to HD, potentially preventing toxicity of mHTT while maintaining HTT function. • QRX-704 activates a Caspase-6 resistant splice isoform: HTTΔ12 • Initial biochemical analyses indicate no major change to global protein folding and biochemistry after exon 12 truncation. • HTTΔ12 RNA and protein was detected in YAC128 brains after ICV administration of QRX-704. • Administration did not cause overt astrogliosis up to 28 days. Scan the code for a digital copy Encephalon A Consortium of ProQR, KTH and UGA ND42223 HD mature iPSC neural culture MAP2 DAPI GFAP 10 25 50 100 200 0 5 10 15 20 25 Droplet digital dPCR [QRX-704 transfected] (nM) HTT ∆12 mRNA (% of total HTT) QRX-704A QRX-704B QRX-704C Figure 3A. COS7 cells MAB2166 (N-terminal) 8A4 (C-terminal) Striatum Figure 8. Western blot, cortex
Transcript of QRX-704, a novel antisense oligonucleotide therapy ... · Institute of Science and Technology...
In vivo evaluation of QRX-704 activityDose-response evaluation of QRX-704 activity in YAC128 HD model administered by intracerebroventricular injection, assessed by droplet digital PCR (Figure 6A). Biodistribution and astrocytosis was assessed in CD1 WT mice by fluorescent in situ hybridization (Figure 6B) and immunohistochemical staining for GFAP (Figure 6C).
Generation of exon 12 isoform-specific monoclonal and polyclonal antibodies, directed against peptides for wt exon 12 or truncated Δ12 (Figure 7A), tested by western blot analysis (Figure 7B).
QRX-704 was administered to FVB-YAC128 mice and sacrificed after 14 days for protein analysis, using crude antibody supernatant, which allowed detecting HTT Δ12 protein (Figure 8).
50100
200400 50
100200
400 50100
200400 50
100200
400 50100
200400
0
20
40
60
Droplet digital PCR
Dose (�g)
HTT
�12
mRN
A(%
ofto
talH
TT) Scrambled
QRX-704Cortex Striatum Hippocampus CerebellumMidbrain
Figure 6A. FVB-YAC128, 2 weeks after single ICV administration
Figure 6B. CD1 mouse 14 days after ICV-administration of QRX-704 (400 µg)
QRX-704-FISH DAPI
1 mm 10 µm
Figure 6C. GFAP immunohistochemistry
aCSF 28 days
400 µg 7 days
100 µg 7 days
400 µg 14 days
200 µg 7 days
400 µg 28 days
GFAP DAPI
Figure 7B. COS7 cells overexpressing HTT WTex12/Δ12 isoforms
anti-WTex12 #1187(Rabbit poly)
anti-total HTTMAB2166
anti-Δ12 #26E7(Mouse mono)
anti-total HTTD7F7
Figure 7A. Isoform specific antibodies
Anti-wtEx12
Anti-Δ12
Results (continued)HTT Δ12 is resistant to HTT586 caspase-6 cleavageCaspase-6 cleavage of HTT Δ12 isoform assessed in lysates from COS7 cells expressing cDNA (Figure 3A), or HD fibroblasts transfected with QRX-704 (Figure 3B), followed by incubation with recombinant active caspase-6.
HTT Δ12 maintains overall folding and biochemical characteristics of canonical HTTBiochemical analysis of purified 23Q or 78Q WT exon 12 or Δ12 HTT isoforms derived in Sf9 insect cells, assessed by SDS- and native PAGE (Figure 4A), CD spectroscopy (Figure 4B) and PTM analysis (Figure 4C).
HTT WT exon 12 or HTT Δ12 overexpressed in SK-N-SH or COS7 cells indicates normal intracellular localization (Figure 5A), and unfolding/digestion pattern in pulse proteolysis (Figure 5B).
Figure 3B. HD Fibroblasts (CAG40/50)
Anti HTT MAB2166 (N-terminal)
Figure 4C. Post translational modification mapped by mass spectrometry Phosphorylated sites with Mod Score >19 displayed
Figure 4B. CD-spectroscopy
Monomeric protein
-40
-30
-20
-10
0
10
20
30
40
190 200 210 220 230 240 250 260
CD [m
deg] Wavelength [nm]
HTT 23Q WTex12
HTT 23Q Δ12
HTT 78Q WTex12
HTT 78Q Δ12
Figure 4A. PAGE of purified proteins
5 % SDS-PAGE 5 % Native PAGE
Figure 5A. Intracellular localization - SK-N-SH cells overexpressing HTT isoforms
HTT WTex12 HTT Δ12
Anti-HTT Hoechst
Figure 5B. Pulse proteolysis
[Thermolysin] (mg/ml)
Anti-HTT MAB2166
-2 -1 0
0.0
0.5
1.0
Western blot quantification
Log [Thermolysin] (mg/ml)
Full
leng
thH
TT(a
.u.)
HTT WTex12 HTT ∆12
Introduction QRX-704 is a novel antisense oligonucleotide-based therapeutic approach, aiming to mitigate mutant Huntingtin (mHTT) toxicity, while maintaining physiological HTT function. Proteolytic cleavage of mHTT generates toxic N-terminal fragments that are hypothesized to be the main contributor to the pathogenesis of Huntington disease (HD). These fragments are formed from a cascade of proteolytic cleavages, initiated by caspase-6 cleavage at position D586. Importantly, HTT586 cleavage is required for HD-like phenotypes in the YAC128 mouse model1. Moreover, mHTT cleavage in turn increases caspase-6 activity, driving pathology in a toxic forward-feedback loop2 (Figure 1A). While toxicity stemming from mHTT cleavage is the primary pathogenic mechanism, data indicates that HTT loss-of-function exacerbates pathology3. In addition, maintaining wild type HTT function may be critical for safety of HTT-targeted therapeutics when treating patients for a long period of time.
Material and MethodsQRX-704 splice-switching activity was tested and optimized in cultured HD fibroblasts (GM04857) and HD iPSC-derived mature neural cultures (ND42223), both from Coriell Institute, assessed by droplet digital PCR and western blotting. Biophysical and biochemical characterization of HTT Δ12 was performed using purified protein, with caspase-6 cleavage assays, CD-spectroscopy, and post-translational modification mapping. Intracerebroventricular (ICV) bolus administration of QRX-704 in in wild type and YAC128 mice, was used to study pharmacodynamic activity, quantified by droplet digital PCR, biodistribution assessed by fluorescent in situ hybridization, reactive gliosis was studied by immunohistochemistry. To better quantify HTT Δ12 protein, exon 12 isoform-specific antibodies were developed and used to detect HTT Δ12 in YAC128 brain tissue.
Results QRX-704 sequence optimization in HD fibroblasts and iPSC-neuronsOligonucleotide lead candidate sequences screened and optimized in HD fibroblasts, assessed by droplet digital PCR and western blot (Figure 2A). Results were verified in HD iPSC neural culture (Figure 2B).
Figure 1A. Caspase-6 cleavage of mHTT drives generation of toxic small N-terminal fragments in forward feedback loop
Figure 1B. QRX-704 activates alternative exon 12 splice isoform, removing HTT586 caspase-6 site
ObjectivesQRX-704 functions at the pre-mRNA level by activating an alternative HTT splice-isoform (HTT Δ12), removing the critical HTT586 caspase-6 cleavage site, thus preventing formation of toxic N-terminal fragments4 (Figure 1B). The aim of the study is to pharmacologically characterize QRX-704 for preclinical development, and describe the novel HTT Δ12 isoform.
Figure 2A. RNA modulation in HD fibroblasts
Ladder Nontransfected QRX-704
0 8 16 24 320
20
40
60
Droplet digital dPCR
[QRX-704 transfected] (nM)
HTT
∆12
mRN
A(%
ofto
talH
TT)
QRX-704A
QRX-704B
QRX-704C
Δ12
WT exon 12
Δ12WT exon 12
Non-transfected
QRX-704A (nM)
8 16 32
QRX-704B (nM)
8 16 32
QRX-704C (nM)
8 16 32
Huntingtin (MAB2166)
Figure 2B. RNA modulation in HD iPSC neurons
QRX-704, a novel antisense oligonucleotide therapy designed to prevent HD pathology while maintaining HTT functionPontus Klein1, Zhana Karneva1, Lodewijk Toonen2, Hyeongju Kim3, Nicholas Caron4, Linda van der Graaf2, Levi Buil1, Geert van der Horst1, Herma Anthonijsz1, Frits van der Ham1, Wouter Beumer1, Michael Hayden4, Ji-Joon Song3, Willeke van Roon-Mom2, Gerard Platenburg1
(1) ProQR Therapeutics, Zernikedreef 9, 2333 CK Leiden, The Netherlands, (2) Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2300RC Leiden, The Netherlands, (3) Department of Biological Sciences, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon 34141, Korea, (4) Centre for Molecular Medicine and Therapeutics (CMMT), CFRI, Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4 Canada
Literature• Graham, Rona K., et al. “Cleavage at the caspase-6 site is required for neuronal dysfunction and
degeneration due to mutant huntingtin.” Cell 125.6 (2006): 1179-1191• Graham, Rona K., et al. “Cleavage at the 586 amino acid caspase-6 site in mutant huntingtin influences
caspase-6 activation in vivo.” Journal of Neuroscience 30.45 (2010): 15019-15029.• Bečanović, Kristina, et al. “A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic
modifier of Huntington disease.” Nature neuroscience 18.6 (2015): 807.• Evers, Melvin M., et al. “Preventing formation of toxic N-terminal huntingtin fragments through antisense
oligonucleotide-mediated protein modification.” Nucleic acid therapeutics 24.1 (2014): 4-12.
SEPTEMBER 2018
ConclusionQRX-704 constitutes a novel therapeutic approach to HD, potentially preventing toxicity of mHTT while maintaining HTT function. • QRX-704 activates a Caspase-6 resistant splice isoform: HTTΔ12 • Initial biochemical analyses indicate no major change to global protein
folding and biochemistry after exon 12 truncation.• HTTΔ12 RNA and protein was detected in YAC128 brains after ICV
administration of QRX-704.• Administration did not cause overt astrogliosis up to 28 days.
Scan the code for a digital copy
EncephalonA Consortium of ProQR, KTH and UGA
ND42223 HD mature iPSC neural culture
MAP2 DAPIGFAP10 25 50 100 200
0
5
10
15
20
25
Droplet digital dPCR
[QRX-704 transfected] (nM)
HTT
∆12
mRN
A(%
ofto
talH
TT)
QRX-704A
QRX-704B
QRX-704C
Figure 3A. COS7 cells
MAB2166 (N-terminal) 8A4 (C-terminal)
Striatum
Figure 8. Western blot, cortex
