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Pathology of antisense oligonucleotides_Summary
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Pathology of antisense oligonucleotides
Cécile Sobry, DVM, DACVP
Department of Pathology, CiToxLAB, Evreux, France
(cecile.sobry@fr.citoxlab.com)
INDICATION OF USE, MECHANISM OF ACTION, CHEMICAL STRUCTURES
Antisense oligonucleotides (AONs) are single stranded, synthetic strands of deoxy- or
ribonucleotide sequences, usually 18-21 nucleotides in length, complementary to the mRNA
sequence of the target gene. These macromolecules afford a unique opportunity to treat
disease at the molecular level.
Indication of use
The potential applications of AONs are numerous because mRNA is ubiquitous and more
readily accessible to manipulation than DNA. Currently, numerous AONs are being evaluated
in clinical trials for treating cancer, inflammation (allergic, autoimmune and some other
inflammatory diseases), metabolic diseases (diabetes, high cholesterol), neuromuscular
disorders, or viral diseases. Although many AONs targeting several genes have been in
clinical trials for many years, only one of these systemically administered AONs,
mipomersen, indicated for homozygous familial hypercholesterolemia, has received
marketing approval from regulatory bodies.
Mechanism
The mechanism by which AONs exert their effects largely depends on their structure and
chemistry. AONs are designed to modulate the expression of proteins encoded by the mRNA,
by binding and interfering with the function of target mRNA. Prior to protein synthesis, premRNA undergoes a range of essential processing steps which are highly regulated, such as 5’capping, polyadenylation, intron-exon splicing, nuclear export, cytoplasmic stabilization, and
ribosomal binding. By interfering with any one of these processing steps, AONs can produce
inhibitory effects. The modulation of splicing by AONs can also be used for the correction of
aberrant splicing and restoration of a functional protein. Another potential mechanism of
AONs is the knockdown of proteins by the activation of the RNAse H enzyme which is a
ubiquitously expressed endonuclease. The cleavage leaves AONs intact and free to bind to
another copy of mRNA. This recycling of AONs makes their effect long lasting, making it
possible to use them in micro or nano molar concentrations (Mansoor and Melendez, 2008).
Chemical structures
Initial studies with AONs, which utilized deoxyribonucleotides, were disappointing, as the
potential therapeutic effect was limited by poor solubility of oligonucleotides, weak
permeation across biological membranes and rapid degradation by endo- and exo-nucleases.
Several chemical modifications were made to improve their stability, potency and
bioavailability. AONs can now be broadly classified into 3 generations. First-generation
AONs have an altered phosphate backbone, most commonly phosphorothioate. Secondgeneration AONs with modified sugars (methyl or methoxyethyl modifications) were
developed to improve the binding affinity and hybridization stability with target mRNA, and
to increase the nuclease resistance. Third-generation AONs include non-ribose based
nucleotides, such as peptide nucleic acids and morpholino phosphorodiamidates. Besides
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modifications in the backbone of the AON, modified bases can be introduced to modify its
characteristics.
This review will focus on the pathological consequences of the pro-inflammatory activity of
AONs and their accumulation in multiple tissues. Specific focus is given to vascular injury
and renal lesions.
TISSUE ACCUMULATION
Accumulation of cytoplasmic granules in macrophages and some epithelial cells from a wide
range of organs and tissues is a class effect observed in all species. Ultrastructural studies and
immunohistochemical staining reveal that the granular material represents the test compound
or associated material contained within endosomal or lysosomal vesicles or vacuoles
(Monteith et al., 1999).
These granules are generally basophilic. Macrophages are enlarged, and have a granular or
foamy appearance. These granular/foamy macrophages are obvious in lymphoid tissues
(lymph nodes, spleen, bone marrow) and liver (Kupffer cells) but can be detected in virtually
all tissues. For AONs given by the subcutaneous route, accumulation is evident at the
injection sites. Granules are also evident in some epithelial cells, principally proximal tubular
cells in the kidney, and at high-doses in hepatocytes or adrenal cortical cells. The degree of
granule accumulation is dose-related. As a consequence of this accumulation, liver, kidney
and lymph nodes enlargement with increased kidney and liver weights are generally observed
after chronic administration. Granules accumulation is slowly reversible and can persist in
tissues for prolonged periods after cessation of the drug.
Some associated degenerative changes such as single cell necrosis of hepatocytes and renal
tubular cells can be observed after administration of high-doses of AONs. In the liver, they
may be related to accumulation of AONs in phagolysosomes but are more probably secondary
to elevated concentrations of cytokines released from activated Kupffer cells. Renal effects
are detailed in a further section.
PRO-INFLAMMATORY EFFECTS
One of the most common effects associated with AON administration is the stimulation of a
pro-inflammatory reaction. The potency of the pro-inflammatory effects is influenced by
AON sequence, base modifications, and backbone chemistry. The phosphorothioate backbone
is known to have some immunostimulatory activity independent of the oligonucleotide
sequence (Hartmann et al., 1996). The basis for the sequence specificity are sequence motifs
in the AONs that are recognized by pathogen-associated molecular pattern (PAMP) receptors
of the innate immune system to activate B cells, monocytes, macrophages, and dendritic cells.
The result of cellular activation is the release of chemokines and cytokines such as MCP-1
and subsequent, dose-dependent increase in lymphoid organ weight, lymphoid hyperplasia
and multi-organ lymphohistiocytic cell infiltrates (Henry et al., 2008).
Vascular injury in monkeys
In monkeys only, vascular lesions consisting of perivascular inflammation and intimal
thickening of variably-sized arteries are occasionally observed with some AONs. Incidence
and severity are not always dose-related, reflecting some individual susceptibility. Affected
animals have generally multiple vascular lesions, particularly in the heart, aorta, kidney, liver,
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lungs, and gastro-intestinal tract. Perivascular inflammation is characterized by mononuclear
or mixed inflammatory infiltrate including eosinophils, with fibroblasts in the adventitia and
perivascular area. The intima is thickened by pale basophilic material and inflammatory cells
with variable cell debris. The endothelium of affected arteries is often hypertrophied and
bulging in the vascular lumen, with cell debris and transmural inflammatory cells. The media
is occasionally affected by degeneration or thickening. In the heart, inflammation and
thickening can also involve the endocardium, particularly along the valves.
Vascular injury has only been observed in monkeys with no similar effect seen in rodents or
in man. It is likely a result of the direct activation of the complement cascade to which
monkeys are particularly sensitive. Complement activation has been demonstrated as the
source of hemodynamic changes reported in monkeys treated with rapid infusion of AONs
(Henry et al., 2002), and has also been observed with slower infusions and with other routes
of drug administration, including subcutaneous dosing (Marquis and Grindel, 2000). Increases
in complement fractions (Bb, C3a) and a subsequent decrease in total complement activity are
generally demonstrated in monkeys with vascular injury.
Long term studies in rodents
The pro-inflammatory activity of AON increases the potential for tumor formation with life
time administration (Levin and Henry, 2008). This has been seen with mipomersen with
increased incidence of fibrohistiocytic tumors in the skin/subcutaneous tissue in rats and mice
(FDA, 2012).
RENAL LESIONS
Renal lesions are a consequence of both tissue accumulation and pro-inflammatory activity
and are seen both in the tubules and in the glomeruli.
Tubular degenerative changes
In addition to cytoplasmic granules, degenerative tubular changes including single cell
necrosis or overt cellular necrosis can be observed in the kidney after administration of
AONs, particularly at high doses. In rats, a worsening of chronic progressive nephropathy was
described with mipomersen (FDA, 2012). Renal tubular degeneration is considered to be
secondary to the sequestration of high concentrations of AONs in phagolysosomes, similar to
the accumulation of β2-microglobulin in rats for instance. This tendency of AONs to
concentrate in the kidney is consistent across species (mice, rats and monkeys), and is
independent of the AON sequence. It is hypothesized that the AONs are filtered by the
glomerulus and subsequently reabsorbed from the tubular lumen into proximal tubular cells
(Henry et al., 2008).
Renal amyloidosis in mice
Degenerative renal tubular changes are generally mild and reversible with AONs. However
some severe, non-reversible lesions can be observed in mice after chronic administration of
high dose-levels, in association with interstitial amyloidosis and consequential papillary
necrosis. Amyloidosis is a common feature in aged mice and it is likely that treatment with a
pro-inflammatory AON speeds the onset and increases the severity of this condition in mice.
The renal papillary necrosis occasionally observed in mice after chronic administration of
AONs at high-doses is probably related to interstitial amyloidosis (Maronpot et al., 1999).
Papillary necrosis is secondary to local ischemia associated with decreased papillary blood
supply due to the tubular dilatation and interstitial deposition of amyloid material in the inner
stripe of the outer medulla of the mouse kidney. Tubular degenerative changes in association
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with renal amyloidosis could be related to the obstruction that follows the papillary injury in
addition to effects of lysosomal accumulation of granular material.
Glomerular lesions
Glomerular lesions can be observed after chronic administration of some AONS. In mice,
lesions are consistent with hyaline glomerulopathy (Wojcinski, 1991), with accumulation of a
homogenous, eosinophilic material in the glomerular tufts and normal or decreased cellularity.
In monkeys, lesions are characterized by enlarged glomeruli, variably increased cellularity of
the tufts and increased mesangium, and occasional infiltration of inflammatory cells.
Identification of antibody-related deposits in mice and complement fragments in monkeys
using both immunohistochemistry and electron microscopy is highly suggestive of an
immune-mediated pathogenesis. Mice and monkeys are more sensitive to oligonucleotideinduced inflammatory effects than man.
References
. Federal Drug Administration (2012) Briefing document, NDA 203568, Mipomersen Sodium
Injection 200 mg/mL. Endocrinologic and metabolic drugs advisory committee meeting.
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enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on
phosphorothioate modification and reversal by heparin. Mol Med 2: 429-438.
. Henry S.P., Beattie G., Yeh G., Chappel A., Giclas P., Mortari A., Jagels M.A., Kornburst
D.J., Levin A. A. (2002) Complement activation is responsible for acute toxicities in rhesus
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