Investigative study of glomerular effects of antisense oligonucleotides in mice

Victoria Castillo

Rationale and History

Antisense oligonucleotides (ODNs) are a class of therapeutic agents designed to inhibit expression of specific genomic and viral RNA targets

Over the past several years, these drugs have shown potential for broad therapeutic applicability

Newer generation ODNs developed in the last few years have a slightly different tox profile than earlier 2nd gen or 1st gen ODN’s

Tend to have stereotypic toxicologic responses in mice, rats, dogs and monkeys regardless of target or sequence

Most prominent features are basophilic granules within kidney (PCTs and rarely glomeruli), liver (kupffer cells, less commonly hepatocytes), lymph nodes, adrenal cortex and rarely pituitary

In mice at high doses in studies of > 6months, can see glomerulopathy that includes amyloid accumulation

– This lesion may be progressive and irreversible in mice over time

Rationale and History

In CD-1 mice, spontaneous amyloidosis is common, but with ODNs: – Increased incidence and severity with time even in the off-

treatment period

In studies of 1 month or less in mice, glomerular changes are not seen

Similar glomerular changes in mice noted with other generation 2.5 oligonucleotide therapies with completely separate sequence and indication

Glomerular changes are dose limiting for chronic studies

Kidney sections were taken from an ongoing mouse study of 56 days duration (10 doses) from control and high (300 mg/kg) dose groups

– This time period for necropsy was fortuitous because it might show the earliest glomerular changes

– Kidney samples in FFPE, OCT and paraformaldehyde

Assessments

Routine microscopy and special stains

Glomerular immunohistochemistry

CD 68 immunohistochemistry

Electron microscopy

Taqman array panel of inflammatory markers

Histology Results

Minimal to mild basophilic granules within the proximal convoluted tubules of all mice given 300 mg/kg/inj

Minimal to mild glomerular changes noted in 6/22 animals given 300 mg/kg which were characterized by:

– Slight increase in mesangial matrix – Increase in glomerular cellularity with occasional inflammatory

cells or nuclear debris – Rare intraglomerular basophilic granules within scattered

glomeruli.

Glomeruli were negative for amyloid by Congo red stains

Negative for fibrosis/collagen deposition by Masson’s Trichrome stain.

GMS and PAS stains both demonstrated slightly thickened basement membranes and/or minimally increased mesangial matrix in the glomeruli of 7/22 mice given 300 mg/kg/inj.

Proximal convoluted tubule

300 mg/kg/injControl

Minimal to mild basophilic granules within PCT epithelial cells of all mice given 300 mg/kg/inj

Glomerular changes

Slightly increased mesangial matrix and rare intraglomerular basophilic granules after 56 days, 300 mg/kg/inj

A- H&E with rare basophilic granules (arrows); B and C – GMS and PAS with mildly increased mesangial matrix

A B C

Results of Immunohistochemical Stains

8/22 mice given 300 mg/kg/inj (high dose) were evaluated by IHC:– von Willebrand’s Factor (vWF, endothelial cell specific)– CD68 (monocyte and macrophage protein specific)– Nephrin and synaptopodin (podocyte specific)

5/8 kidneys from high dose group had increased vWF immunostaining in glomeruli

All had markedly increased CD68 immunostaining in treated mouse kidneys

No differences for nephrin and synaptopodin staining between controls and treated

Immunostaining for von Willebrand’s Factor

Areas of increased staining within glomerular tufts could represent areas of endothelial hypertrophy, degeneration, and/or accumulation of vWF protein within cytoplasmic spaces

Control 300 mg/kg/inj

Immunostaining for CD68

Control 300 mg/kg/inj

Increase in CD68 immunostaining suggests cytokine protein upregulation

Electron Microscopy Results in Treated Mice

Thickened and irregular endothelial cell linings, but with fenestrations maintained

Endothelial cells were hypertrophied with expanded cytoplasm and/or pyknotic nuclei

Numerous capillaries contained cellular debris, amorphous granular material, and vesicles.

Glomerular basement membrane changes– longitudinal “layering” or “splitting”– electron-dense and electron-lucent deposits– thickening

Endothelial cells, mesangial cells, and podocytes often had numerous membrane-bound vesicles containing electron-dense material.

Mesangial matrix was mildly thickened and contained rare electron-dense deposits and cellular debris.

Proximal Convoluted Tubule Changes

Control 300 mg/kg/inj

Electron-dense material (drug?) in lysosomes

Glomerular Changes

Numerous lysosomes containing electron dense material in endothelial cells, podocytes, and mesangial cells.

300 mg/kg/injControl

Glomerulus - Endothelial Cell Changes

Control 300 mg/kg/inj

Capillary lumens contained cellular debris, granular material, and vesicles.Many endothelial cells appeared hypertrophic and had thickened, irregular cell processes.

Glomerulus - Endothelial Cell Changes

300 mg/kg/inj 300 mg/kg/inj

Thickened, irregularly branching endothelial cell processes (arrows) and basement membrane splitting (arrowheads).

Glomerular Endothelial Cell and Basement Membrane Changes

300 mg/kg/inj 300 mg/kg/inj

Thickened, branching endothelial cell processes (arrows)

Splitting (arrowheads) and thickening of glomerular basement membrane

Taqman® Gene Signature Mouse Immune Array

Kidney samples from 6 controls, 6 high dose treated with histologic evidence of glomerulopathy and 3 high dose treated with no histologic evidence of glomerulopathy were analyzed using the Taqman® Gene Signature Mouse Immune Array to assess possible immune and complement pathways affecting glomerular injury after treatment.

Each card contains 96 TaqMan® Gene Expression Assays for quantitative analysis of targets known to have implications in immune response

– The targets include cytokines, chemokines, growth factors, immune regulators, apoptosis markers, ischemia markers, tissue-specific markers, and others including classic endogenous markers.

The Taqman® raw data was evaluated using RQ Manager Software and assessed using the Data Assist ™ Software

– The data was initially filtered by excluding fold change <2 to establish statistical significance

Taqman® Gene Signature Mouse Immune Array Fold ChangesNormalized to 18s and β actin and compared to controls

This is a representation of selected genes of the 29 found to be statistically significant in the upregulation of mouse immune response

Assay Type

Evidence of glomerular lesions in treated mice (Fold

change)

No evidence of glomerular lesions

in treated mice (Fold change)

Il10 Target 110.77 70.60

Ccl3 Target 24.72 13.17

Ccl2 Target 20.45 18.23

Il6 Target 10.11 5.54

Il1a Target 7.55 7.64

Gzmb Target 6.58 4.10

Cxcl10 Target 6.41 7.48

Cd68 Target 6.10 5.43

Taqman® Gene Signature Mouse Immune Array Fold Change (normalized and compared to controls)

The genomic microarray data indicates there is marked cytokine and proinflammatory upregulation in kidneys of treated mice as compared to controls, with the mice most

histologically affected showing slightly greater expression than those without histologic lesions

Discussion

ODNs are highly renally excreted and that due to accumulation within the proximal tubule cell lysosomes, renal tissue levels of ODNs may exceed that of other organs

ODNs are known to have strong proinflammatory activity, and localization within renal tubular or glomerular lysosomes may be strong local stimulus

Initial glomerular changes of basement membrane deposits and BM splitting are suggestive of an immune-mediated pathogenesis

Initial site of injury at day 56 is the endocapillary endothelial cell – potential target for circulating cytokine mediated injury – whereas podocytes (which appeared unaffected) more often represent  targets of

direct cytotoxicity from the filtrate.

The deposits and denuded areas around the capillary tufts may provide a nidus for deposition of amyloid fibrils, hyaline material and/or collagen over time. Immunomodulatory pathophysiologic mechanism is supported by upregulation of immune cytokines by microarray and positive CD68 staining.

Many of the upregulated genes in this study, including CD68, have also been implicated in amyloid formation via induction of the SAA pathway

– suggests that increased amyloid formation chronically may also be related to this proinflammatory activity

Summary

After 56 days and 10 doses of an ODN, changes were noted microscopically and ultrastructurally in the glomeruli of mice kidneys in association with the presence of basophilic granules in adjacent tubules and in some glomeruli.

Glomerular endothelial cells appear to be the initial target, and were accompanied by ultrastructural changes in the associated basement membrane; similar changes have been noted in association with an immune pathogenesis within glomeruli

Using Taqman® gene expression assay, marked changes in proinflammatory genes were present in high dose treated mice as compared to controls

Marked CD68 immunopositivity in treated mouse kidneys correlated with increased CD68 mRNA in the genomic data, indicative of inflammatory cytokine upregulation at both proteomic as well as genomic levels

Together the data suggest that immunomodulatory effects of this ODN are linked mechanistically to development of glomerulopathy and later amyloidosis in mice

Acknowledgement

Tracy Gales

David Mullins

Ken Frazier

Rosanna Mirabile

Janice Kane

Bobbi Thomas

Marshall Scicchitano

Lauren Tierney

Patty Pesavento