review of galnac-sirna conjugates across multiple …review of galnac-sirna conjugates across...

Review of GalNAc-siRNA Conjugates Across Multiple Programs Demonstrates No Evidence of Thrombocytopenia or Pro-inflammatory EffectsKristina Perry, Julia Hettinger, Brenda Carito, Natalie Keirstead, Michael PlackeAlnylam Pharmaceuticals, Inc., Cambridge, MA, USA

AbstractCoagulation dysfunction, predominantly mediated by thrombocytopenia and immunostimulatory effects, including complement activation, has been associated with a number of oligonucleotides with high phosphorothioate (PS) content, with most reports associated with high PS antisense oligonucleotides (ASOs). These effects include lymphoid hyperplasia, lymphohistiocytic infiltrates in multiple organs, immune-mediated glomerulonephritis and vasculitis. Further, moderate to severe thrombocytopenias have been reported both nonclinically and in humans that may also be immune-mediated, which often results in severe bleeding disorders.GalNAc-siRNA conjugates are characterized by low PS content. Here we describe the safety profiles of GalNAc-siRNAs in rats and non-human primates (NHP) given siRNA conjugates in toxicology studies as repeat doses up to 13 weeks and under chronic dosing conditions (26 weeks for rats and 39 weeks for NHP). All of these studies showed an absence of any platform-wide pro-inflammatory changes or evidence of thrombocytopenia or diminished platelet function. This review of nonclinical data from eight GalNAc-siRNA conjugates revealed no significant effects on hematology parameters, including platelets, nor other parameters that indicate pro-inflammatory or coagulation liabilities (cytokines, CBC values and coagulation parameters, and microscopic evaluations of relevant tissues). In summary, the lack of immunostimulatory effects of GalNAc-siRNA conjugates strengthens the hypothesis that high PS content is responsible for the pro-inflammatory mechanisms and thrombocytopenia observed with unconjugated ASOs, compounds with wider tissue distribution and longer plasma half-life compared to GalNAc-siRNA conjugates, which are rapidly cleared from plasma and predominantly distributed to the intended target organ (liver).

Summary• Eight low PS GalNAc-siRNAs were evaluated for immunostimulatory properties and potential to produce thrombocytopenia.

• No effects on platelet numbers or immune stimulation were observed in rats or NHPs across programs: – There were no platform-wide siRNA conjugate effects on hematology or coagulation parameters: APTT, platelet numbers, leukocyte counts were all within normal limits. – There were no positive signals of immune modulation in mouse or NHP cytokine assays. – There was no macroscopic or microscopic evidence of immune stimulation by siRNAs: spleen weights were normal, and siRNAs were not associated with spleen or lymph

node lymphoid hyperplasia, immune-mediated glomerulonephritis, nor vasculitis.

• We hypothesize that while the low PS content remains effective in improving stability of double-stranded siRNAs, it does not lead to the degree of protein binding and increased plasma half life that may be responsible for the observed immune and coagulation effects observed with fully PS single-stranded antisense oligonucleotides.

AcknowledgmentsWe thank Dr. Carole Harbison, Dr. Maja Janas and Kellie Sawyer for technical expertise and data review.

References1. Havens, M.A. et al. 2016. Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Res. 44(14):6549-63.

2. Moreno, P.M. et al. 2014. Therapeutic antisense oligonucleotides against cancer: hurdling to the clinic. Front Chem. 2:87.

3. Shen, W. et al. 2015. 2’-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF. Nucleic Acids Res. 43(9):4569-78.

4. Bennett, C.F. et al. 2010. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol. 50:259-93.

5. Henry, S.P. et al. 1997. Inhibition of coagulation by a phosphorothioate oligonucleotide. Antisense Nucleic Acid Drug Dev. 7(5):503-10.

6. Senn, J.J. et al. 2005. Non-CpG-containing antisense 2’-methoxyethyl oligonucleotides activate a proinflammatory response independent of Toll-like receptor 9 or myeloid differentiation factor 88. Pharmacol Exp Ther. 314(3):972-9.

7. Ferdinandi, E.S. et al. 2010. Preclinical toxicity and toxicokinetics of GTI-2040, a phosphorothioate oligonucleotide targeting ribonucleotide reductase R2. Cancer Chemother Pharmacol. 2011 Jul;68(1):193-205.

8. Frazier, K.S. et al. 2014. Species-specific inflammatory responses as a primary component for the development of glomerular lesions in mice and monkeys following chronic administration of a second-generation antisense oligonucleotide. Toxicol Pathol. 42(5): 923-935.

Introduction• Therapeutic oligonucleotides are chemically modified to prevent nuclease degradation and to improve stability and binding affinity. (Havens 2016, Moreno 2014)• The phosphorothioate (PS) backbone modification replaces a non-bridging oxygen atom with a sulfur atom.

Phosphodiester Linkage (PO)

PhosphorothioateLinkage (PS)

• PS modification is commonly used because it decreases nuclease degradation and increases binding of the oligonucleotide to plasma proteins which extends half-life in plasma. (Shen 2015) (Bennet 2010)

• Oligonucleotides with a fully PS backbone have characteristic toxicities such as increased coagulation times, decreased platelets, and immune activation. (Henry 1997, Moreno 2014, Senn 2005, Ferdinandi 2011)

Nucleotide

• Effects of low PS GalNAc-siRNAs on these pathways have not been reported.

Nucleotide

• The goal of the current study is to review key data from preclinical studies, focusing on rat and NHP sub-chronic and chronic GLP (good laboratory practice) studies, comparing key hematology and cytokine parameters related to coagulation and immune stimulation from eight GalNAc-siRNA conjugates with different sequences and mRNA targets.

No Evidence of Thrombocytopenia in Rats or NHPs Across Programs

SD Male Rats - Subchronic GLP

03 /µL)

AT3 HBVAS1 CC5 TTRGO1 PCSAAT

SD Female Rats - Subchronic GLP

03 /µL)

NHP Male - Subchronic GLP

03 /µL)

NHP Female - Subchronic GLP

03 /µL)

SD Male Rats - Chronic GLP

03 /µL)

AT3AS1 CC5AAT

SD Female Rats - Chronic GLP

03 /µL)

AT3AS1 CC5AAT

NHP Male - Chronic GLP

03 /µL)

AT3AS1 CC5AAT

NHP Female - Chronic GLP

03 /µL)

AT3AS1 CC5AAT

Figure 1. Platelet counts from eight sub-chronic studies (7-14 week) in male and female Sprague Dawley rats (A and B), and four chronic studies (26 week) in male and female Sprague Dawley rats (C and D), with the same compounds evaluated 7-14 weeks in male and female NHPs (E and F), or 39-41 weeks in male and female NHPs (G and H). The dose groups are listed on the x-axis and platelet (PLT) count in cells x103/μL on the y-axis. Dosing frequency was typically once weekly (QW). Low doses ranged from 5-30 mg/kg across programs, with Mid doses of 30-150 mg/kg, and High doses of 50-300 mg/kg. The AT3 program was an exception, with doses ≤3 mg/kg QW due to exaggerated on-target pharmacology. The dotted lines are age-, sex-, and species-specific reference intervals obtained from the CROs (contract research organizations). All platelet values remain within normal reference ranges, with no evidence of thrombocytopenia, following up to 26-39 weeks of chronic dosing of up to 300 mg/kg QW GalNAc-siRNA conjugates.

No Platform-Wide Effects on Neutrophil Counts

SD Male Rats - Subchronic GLP

(103 /µ

SD Female Rats - Subchronic GLP

(103 /µ

NHP Male - Subchronic GLP

(103 /µ

L)AT3 HBVAS1 CC5 TTRGO1 PCSAAT

NHP Female - Subchronic GLP

(103 /µ

SD Male Rats - Chronic GLP

(103 /µ

AT3AS1 CC5AAT

SD Female Rats - Chronic GLP

(103 /µ

AT3AS1 CC5AAT

NHP Male - Chronic GLP

(103 /µ

AT3AS1 CC5AAT

NHP Female - Chronic GLP

(103 /µ

AT3AS1 CC5AAT

Figure 3. Neutrophil counts from eight sub-chronic studies (7-14 week) in male and female Sprague Dawley rats (A and B), and four chronic studies (26 week) in male and female Sprague Dawley rats (C and D), with the same compounds evaluated 7-14 weeks in male and female NHPs (E and F), or 39-41 weeks in male and female NHPs (G and H). The dose groups are listed on the x-axis and neutrophil (NEUT) count in cells x103/μL on the y-axis. Dosing frequency was typically once weekly (QW). Low doses ranged from 5-30 mg/kg across programs, with Mid doses of 30-150 mg/kg, and High doses of 50-300 mg/kg. The AT3 program was an exception, with doses ≤3 mg/kg QW due to exaggerated on-target pharmacology. The dotted lines are age-, sex-, and species-specific reference intervals obtained from the CROs. There was a minimal to mild neutrophilia across dose groups and studies in the CC5 program only, that was expected and secondary to on-target pharmacology. There were no siRNA-related effects on lymphocytes or monocytes across programs (not shown).

No Platform-Wide Effects on Coagulation Parameters (APTT)

SD Rats - Subchronic GLP

NHP - Subchronic GLP

SD Rats - Chronic GLP

AT3AS1 CC5AAT

NHP - Chronic GLP

AT3AS1 CC5AAT

SD Rats - Subchronic GLP

NHP - Subchronic GLP

SD Rats - Chronic GLP

AT3AS1 CC5AAT

NHP - Chronic GLP

AT3AS1 CC5AAT

Figure 2. Activated partial thromboplastin time (APTT) from eight sub-chronic studies (7-14 week) in Sprague Dawley rats (A), and NHPs (B), and four chronic studies (26 week) in Sprague Dawley rats (C), or 39-41 weeks in NHPs (D). Male and female data have been combined for each dose group as APTT was very consistent between sexes. The dose groups are listed on the x-axis and APTT in seconds on the y-axis. Dosing frequency was typically once weekly (QW). Low doses ranged from 5-30 mg/kg across programs, with Mid doses of 30-150 mg/kg, and High doses of 50-300 mg/kg. The AT3 program was an exception, with doses ≤ 3 mg/kg QW due to exaggerated on-target pharmacology. The dotted lines are age- and species-appropriate reference intervals obtained from the CROs. There were no siRNA-related effects on APTT, or other coagulation factors (not shown), across programs and studies.

No Test Article-Related Effects on Serum Cytokines in NHP 4 or 24 Hours Post-Dose

IL-6 NHP 4hr

se) AT3AS1 TTRPCSAAT CC5 HBV

MCP-1 NHP 4hr

AT3AS1 TTRPCSAAT

IL-6 NHP 24hr

se) AT3AS1 TTRPCSAAT CC5 HBV

MCP-1 NHP 24hr

AT3AS1 TTRPCSAAT

Figure 4. Serum cytokine levels were measured in seven NHP sub-chronic GLP studies at 4 and 24 hours post-dose on Day 1 (shown) and on the final day of dosing with siRNA (not shown, but similar to Day 1*). Cytokines were measured using Millipore multiplexed cytokine kits (multiplexed bead based detection). The plate was read on the Bio-Plex Suspension Array (Luminex) system. The dose groups are listed on the x-axis and cytokine fold change compared to predose levels on the y-axis. Dosing frequency was typically once weekly (QW). Low doses ranged from 5-30 mg/kg across programs, with Mid doses of 30-150 mg/kg, and High doses of 50-300 mg/kg. The AT3 program was an exception, with doses ≤3 mg/kg QW due to exaggerated on-target pharmacology. The dotted line shows a fold change of 1 (no change from predose). There were no test article-related findings for any of the evaluated cytokines. Cytokine panels varied by CRO, depending upon the cytokines validated for GLP (good laboratory practices). HBV and CC5 cytokine assays were run GLP at the CRO and the remaining programs were run at Alnylam, using similar procedures (Luminex assays). IL-6 is a classic indicator of inflammatory responses from CpG oligonucleotides and MCP-1 has been shown to be increased in fully PS containing oligonucleotides without CpG. There were no cytokine elevations related to siRNA administration, up to doses of 300 mg/kg QW x 14 on Day 1.

*One exception: there was a mild increase in IL-6 in the CC5 program, seen only on Day 30 that was expected and correlated with neutrophilia, and was secondary to on-target pharmacology.

No Test Article-Related Effects on Plasma Complement (Bb and C3a) Concentrations

NHP Complement Bb 4hr Postdose

se) AS1AAT TTRscPCSSC

NHP Complement Bb 24hr Postdose

se) AS1AAT TTRscPCSSC

NHP Complement C3a 4hr Postdose

AS1AAT TTRscPCSSC

NHP Complement C3a 24hr Postdose

AS1AAT TTRscPCSSC

Figure 5. Complement activation from four NHP GLP sub-chronic studies. Complement Bb (A and B) and C3a (C and D) were measured in plasma 4 and 24 hours post dose (Day 1) using Bb and C3a Plus Enzyme Immunoassay Kit (Quidel Corporation). The dose groups are listed on the x-axis and complement fold change compared to predose levels on the y-axis. Dosing frequency was typically once weekly (QW). Low doses ranged from 5-30 mg/kg across programs, with Mid doses of 30-150 mg/kg, and High doses of 50-300 mg/kg. The dotted line shows a fold change of 1 (no change from predose). There were no elevations in complement factors Bb or C3a related to siRNA administration.

Male SD Rats - Subchronic GLP

Female SD Rats - Subchronic GLP

Male NHP - Subchronic GLP

Female NHP - Subchronic GLP

No Evidence of Pro-Inflammatory Effect in Spleen Weight

Figure 6. Spleen to brain weight ratios. A. and B. eight sub-chronic studies (7-14 week) in male and female Sprague Dawley rats, C. and D. eight sub-chronic studies (7-14 week) in male and female NHPs. The dose groups are listed on the x-axis and spleen to brain weight ratios (%) on the y-axis. Dosing frequency was typically once weekly (QW). Low doses ranged from 5-30 mg/kg across programs, with Mid doses of 30-150 mg/kg, and High doses of 50-300 mg/kg. The AT3 program was an exception, with doses ≤3 mg/kg QW due to exaggerated on-target pharmacology. There have been no increases in spleen weights (including absolute weights or compared to body weight, not shown) in any studies, demonstrating no evidence of pro-inflammatory effects that can manifest as splenomegaly.

No Evidence of Immune-Mediated Glomerulopathy With siRNA Treatment

Figure 7. Four GalNAc-siRNA conjugates have been evaluated ultrastructurally in rats and/or NHPs. In the kidney, there was no evidence of toxicity and specifically, no immune-mediated glomerulopathy as has been previously reported with ASOs (Frazier et al., 2014). The structure of the glomerular capillary wall is llustrated above, and confirms no evidence of glomerulopathy with siRNA treatment, the glomerular capillary basement membrane is intact and well-organized, with no expansion by immune-deposits or amyloid. Kidney sections from animals administered GalNAc-siRNA conjugates (B) were similar to vehicle controls (A).

Vehicle Control Rat; 16,500X 30 mg/kg Q2D x 6 siRNA in Rat, 16,500x

review of galnac-sirna conjugates across multiple …review of galnac-sirna conjugates across...

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