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Amyloid Cardiomyopathy
27
[84] Mikhael, JR; Schuster, SR; Jimenez-Zepeda, VH; et al.
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In: Amyloidosis ISBN: 978-1-63485-884-7
Editor: Raquel Watts © 2017 Nova Science Publishers, Inc.
Chapter 2
NOVEL THERAPIES FOR AMYLOIDOSIS IN
THE ERA OF RNAI AND IMMUNOTHERAPY
Sandy W. Wong
*
, MD and Raymond L. Comenzo, MD
Departments of Medicine and Pathology and the Division of Hematology-
Oncology, Tufts Medical Center, Boston, MA
ABSTRACT
Abnormal fibrillar deposition of protein causing organ failure
characterizes all forms of amyloidosis. Amyloidosis was uniformly a
rapidly fatal disease without effective treatment options around 20 years
ago. Multiple advances especially in light chain (AL) amyloidosis have
occurred since, first with the advent of melphalan-based autologous stem
cell transplant and then the introduction of bortezomib-based treatment.
Both of these treatment modalities, derived from multiple myeloma
research, kill the clonal cells producing immunoglobulin light chains.
While research continues in search of cytotoxic therapies for AL, current
studies are directed at the production, stability and clearance of light-
chains as well as other types of amyloid proteins. We summarize the
current investigations utilizing RNAi and immunotherapy to target the
amyloid protein itself.
*
Corresponding author: Sandy W. Wong, MD, TEL 617-636-6454,
Swong7@tuftsmedicalcenter.org.
Sandy W. Wong and Raymond L. Comenzo
30
INTRODUCTION
In the 1850s, Rudolph Virchow applied the term amyloid, derived from
the Greek word “amylon” for starch, to waxy tissue deposits in organs from
patients with inflammatory diseases, because the deposits reacted with iodine
and sulfuric acid [1, 2]. He surmised that the deposits consisted of starch-like
substances. Shortly thereafter, Carl Friedreich and August Kekule with protein
staining concluded that the waxy substances were made of protein, not starch
[1, 2]. Nowadays, we know that both scientists were correct and that amyloid
deposits are comprised of both protein and glycosaminoglycans. Although
these observations marked the beginning of amyloid research, most of the
clinical applications of amyloid research have focused on eliminating the
deposits or the factory producing the amyloid proteins. While research
continues in search of other cytotoxic therapies, current studies are directed at
the production, stabilization and clearance of the amyloid protein by utilizing
today’s technologies in immunotherapy, protein stabilization and RNA
interference (RNAi).
AN UNMET NEED
Although great strides have been made in the treatment of AL amyloidosis
with the advent of autologous stem cell transplant (ASCT) [3, 4] and
bortezomib-based therapy [5] to eliminate the cells producing the culprit light
chains, systemic amyloidoses continue to cause mortality and morbidity [6-8].
In the case of AL amyloidosis, only 25% of patients are eligible for ASCT
with a treatment-related mortality of 5-13% [4, 9-11]. A reduction in light
chains through elimination of the neoplastic plasma cells, results in
hematologic remission, improved organ function and better outcomes [5, 12,
13]. A hematologic remission though required for an organ response, does not
necessarily guarantee improved organ function [11]. In a study with AL
patients with cardiac involvement, cardiac biomarker responses at 6 months
occurred in only 57% and 37% of patients who achieved hematologic
complete (CR) and very good partial responses (VGPR) respectively [14]. For
ASCT patients who achieve CR at 1 year, 79% had organ responses [15].
Novel Therapies for Amyloidosis in the Era of RNAi …
31
Addition of bortezomib and dexamethasone as consolidation following ASCT
improved the proportion of patients in CR with an organ response rate of 70%
at two years [16]. In addition, rates of organ response appear to differ
depending on the affected organ [16, 17]. For patients in hematologic CR,
renal and cardiac responses at 1 year differed at 35% and 57% respectively
[16]. While modern cytotoxic therapies have improved hematologic response
rates, organ response rates remain low and end-organ dysfunction remains a
major cause of death [18]. In fact, nearly half of patients die within a year of
diagnosis in AL [19].
Similarly, liver transplantation to replace the hepatocytes in mutant
transthyretin (ATTR) amyloidosis is limited to a very few select patients [20,
21]. The procedure also carries the risk of life-long immunosuppression,
transplant-related death and disease recurrence despite transplantation [22].
Clearly, better therapies to improve end-organ damage are urgently needed for
this orphan disease.
GENERAL BIOLOGY OF AMYLOIDOSIS
Amyloidosis is an organ deposition disease of abnormal fibrillar proteins
which are formed from various distinct precursor proteins. There are now > 25
precursor proteins [23] which can be amyloidogenic. Of patients diagnosed
with amyloidosis, AL comprised 62% of cases, ATTR 25%, and AA 5% [24].
The general biology of amyloid formation consists of a cellular “factory”
which produces an amyloidogenic protein. This protein which is
thermodynamically unstable, misfolds and aggregates to form fibrils. These
fibrils then deposit in organs causing severe organ dysfunction. The source of
amyloid deposits may vary, but all amyloid fibrils are structurally composed of
unbranched fibrils 8-10 nm in diameter with a cross-β pleated sheet
conformation [25, 26]. As such, all types of amyloid can be detected
histologically by interaction with thioflavin T and Congo red [27, 28].
Amyloid fibrils in tissue specimens exist bound to other circulating proteins
such as serum amyloid P component (SAP) and glycosaminoglycans such as
heparan sulfate. The common pathway of amyloid fibril formation and the
universal presence of bound glycosaminoglycans and SAP may allow certain
types of therapeutics targeting amyloid fibrils to become broadly applicable.
Sandy W. Wong and Raymond L. Comenzo
32
TARGETING THE PRODUCTION OF
AMYLOID-FORMING PROTEINS
Fibrillar amyloid deposits are derived from a precursor protein that is
made by a cell. AL amyloid deposits are composed of immunoglobulin light
chains (LC) [29, 30]. Light chain variable region germline gene usage is
restricted, occurs at a lambda:kappa frequency of 4:1 [11], and influences
organ involvement [29, 31-35].
Elimination of the plasma cell has become the main strategy for treatment
of AL amyloidosis by melphalan-based ASCT, proteasome-inhibitors,
immunomodulatory agents and steroids, cytotoxic treatments recently
reviewed elsewhere [36]. Novel therapies in development focus instead on
disrupting the intracellular production of the amyloidogenic light chains. Since
most of the organ damage in amyloidosis is caused by amyloid LC deposits,
not plasma cell burden or proliferation, methods of shutting down production
of LC may prove to be clinically beneficial.
One strategy to reduce light chains is to use RNA interference (RNAi)
technology to knock-down light chain mRNA and hence decrease light chain
protein production from plasma cells. Small interfering RNA (siRNA) is a part
of the RNAi system that bind to mRNA and target it for degradation, thereby
regulating protein translation [37]. Multiple investigators have developed
different siRNAs to LC mRNA. In AL-Base, a database of germline LC genes
in AL patients, 21.1% of the sequences were from the κ1 family (08/018).
Thus three different κ1-directed siRNAs were developed to inhibit synthesis of
light chains in vitro (2 against the variable κ1, 1 against the constant κ1).
These siRNAs significantly reduced local production and circulating levels of
LC in vivo in a κ1-expressing plasmacytoma mouse model of AL [38].
In addition, custom siRNA was created against the V, J or C portions of
λ6 (Wil) and λ2 LC molecules, which produced a reduction in LC secretion
upon transfection in vitro [39]. Both of these studies demonstrate that siRNA
could be produced to λ and κ amyloidogenic LC and could effectively knock-
down LC production. However, the challenge to using these types of siRNAs
includes the lack of broad applicability because of the high specificity of these
reagents for only certain germline genes. For translation into the clinical
setting, siRNA would need to be custom-designed per patient [38] which may
prove to be cost-prohibitive and time-consuming. In fact, siRNA against κ1
could only be used for less than half of kappa AL patients [38].
Novel Therapies for Amyloidosis in the Era of RNAi …
33
To address the obstacle of LC variable region gene sequence diversity, a
siRNA pool targeting the consensus sequences in the λ LC constant region was
developed [40]. This siRNA pool was able to reduce λ LC production and
secretion by human plasma cells in vitro regardless of sequence diversity.
Interestingly knock-down of the λ light chain caused terminal endoplasmic
reticulum stress with resultant apoptosis in AL cell lines and patient plasma
cells making intact immunoglobulin [40]. Furthermore, in a murine xenograft
plasmacytoma model with an AL amyloid cell line, the siRNA pool delivered
by electroporation, resulted in a reduction in λ LC mRNA [41]. Similar results
were observed for a siRNA pool against the consensus sequences in the κ
constant region [41].
RNAi methods to reduce amyloidogenic LC are precise with low
likelihood of off-target effects due to minimal sequence homology between
LC and other genes [38]. However, the delivery method of siRNA requires
further development. The delivery method of siRNA by electroporation done
in mice could not be translated into clinical practice. Optimal delivery of
siRNA to the target cells is an area of active investigation [41-43].
RNAi is also being explored for the treatment of ATTR amyloidosis.
TTR, also known as pre-albumin, circulates as a homotetramer and is made
primarily in the liver with a small amount in the choroid plexus of the brain
and the retina [44]. Wildtype TTR in its monomeric form possesses a
constitutive ability to aggregate into amyloid fibrils [45]. Mutant TTR
occurring as a single point mutation increases the propensity of the TTR
tetramer to dissociate into monomers, misfold and form amyloid fibrils [46].
Depending on the pattern of predominant organ involvement, the amyloid
deposits could result in familial amyloid cardiomyopathy (FAC), familial
amyloid polyneuropathy (FAP) or leptomeningeal amyloidosis [44].
Liver transplantation is a way of halting the production of the
amyloidgenic TTR for hereditary ATTR. Progression of polyneuropathy in
patients with TTR-FAP is reduced by orthotopic liver transplant with
transthyretin homozygous wildtype liver donors [47, 48]. Conversely, a liver
graft from a Val30Met amyloid patient, used as a donor organ in a ‘domino’
transplant’, resulted in development of ATTR amyloidosis in the recipient
[49]. Stopping hepatocyte production of the amyloidogenic TTR may also stop
progression of disease.
The liver is also the organ most efficiently targeted by lipid nanoparticles,
a delivery vehicle for siRNA [43]. RNAi technology delivered by lipid
nanoparticles has demonstrated activity in several disease models [43, 50, 51].
Different formulations of TTR siRNA have been studied recently in multiple
Sandy W. Wong and Raymond L. Comenzo
34
clinical trials. ALN-TTRSc (Revusiran) is a TTR siRNA drug, conjugated to
triantennary N-acetylgalactosamine [43], a ligand which binds to surface
asialoglycoprotein receptor on hepatocytes, thereby targeting the siRNA to the
liver. The siRNA works against both wildtype and mutant TTR mRNA due to
its ability to bind to a conserved sequence in the 3’ untranslated region of TTR
mRNA [52]. In pre-clinical studies, revusiran reduced TTR deposits in
peripheral tissues greater than that achieved with the TTR stabilizer tafamidis
[52]. Furthermore, revusiran compared to TTR antisense oligonucleotide
(ASO) in mice induced more potent knockdown of TTR, despite lower levels
of revusiran in the liver and kidney.
ALN-TTRSc, administered as a subcutaneous injection, was studied in a
first-in-human phase I placebo-controlled clinical trial (NCT01814839) in 85
healthy volunteers [53]. Multiple doses of the drug were well-tolerated except
for mild-to-moderate injection site reactions [52]. Mean reduction in serum
TTR with weekly dosing of the drug was up to 92% [52]. A phase II study
(NCT01981837) with revusiran in TTR cardiac amyloidosis has also been
completed with an open-label extension ongoing. The drug was given to 26
patients (12 age-related ATTRwt, 14 hereditary ATTRmt), administered as 5
daily doses followed by weekly dosing for 2 years. It was well tolerated and
associated with > 85% sustained knockdown of serum TTR [54]. Similar
levels of knockdown were observed for both wildtype and mutant TTR [52].
Tolerance and adverse effects were similar to the phase I trial [52]. A phase III
randomized, double-blind, placebo-controlled study (ENDEAVOUR,
NCT02319005) with revusiran given for 18 months in patients with TTR-FAC
is currently ongoing. A phase II study (NCT02595983) with revusiran in TTR-
FAP patients with worsening disease post-liver transplant is also recruiting
patients [53].
ALN-TTR02 (Patisiran) is a TTR siRNA therapeutic encapsulated in a
lipid nanoparticle with the ability to knockdown both wildtype and mutant
TTR. The drug is administered intravenously, opsonized by apolipoprotein E
and is targeted to the liver via interactions with hepatic ApoE receptors [52,
55]. In a phase I randomized single-dose placebo-controlled trial, 32 ATTR
patients and 17 male healthy volunteers were given this drug [55]. The drug
was well-tolerated except for mild-to-moderate infusion related reactions in
8% of patients. Mean reductions in TTR ranged from 82% to 87% at doses of
0.15-0.3 mg/kg. Reductions of TTR were 57%-67% at 28 days after
administration [55]. Patisiran has also been studied in a phase II dose-
Novel Therapies for Amyloidosis in the Era of RNAi …
35
escalation study in 29 patients with stage I or II TTR-FAP. Maximum mean
reduction in TTR reached 87% for patients in the 0.3mg/kg every 3 week
cohort [52]. Although the majority of adverse effects were mild or moderate, 2
patients had serious adverse effects with 1 discontinuation due to severe
emesis [52]. The open-label extension (NCT01961921) of the phase II trial is
currently ongoing. Preliminary analysis confirmed the safety findings from the
prior trials [52]. Interestingly, neuropathy impairment scores were stable over
12 months compared to worsening scores by historical controls [52]. This drug
is currently in phase III placebo-controlled trial (APOLLO) for a duration of
therapy of 15 months (NCT01960348) with once weekly dosing in TTR-FAP.
Primary endpoint is change from baseline of modified Neuropathy Impairment
Score +7 and change from baseline in the Norfolk Quality of Life Diabetic
Neuropathy questionnaire [44]. Notably, penetration of patisiran into the CNS
system or eye does not occur after systemic administration [55].
Antisense oligonucleotide drugs are another technology being used to
inhibit hepatic TTR expression. ASO are short oligonucleotides which
complement and bind to the mRNA of TTR, resulting in ribonuclease H-
mediated degradation. IONIS-TTR
Rx
is an ASO that can bring about
destruction of both wildtype and mutant TTR [56]. Transgenic mice with
human TTR I84S given this drug showed TTR protein suppression of 80%
[56], which was then confirmed in non-human primate models [57]. In a phase
I dose-escalation study, single doses of IONIS-TTR
Rx
were given to healthy
volunteers. The drug was well tolerated up to 400mg with a favorable safety
profile. There were rapid dose-dependent reductions in circulating TTR levels
of around 80% after 4 weeks [52, 58]. A phase II/III randomized, double-
blind, placebo-controlled trial (NCT01737398) with this drug in stage I or II
TTR-FAP is underway with an open-label extension (NCT02175004) [52].
Like ATTR and unlike AL, the source of the precursor protein in AA
amyloidosis is the liver. The liver produces serum amyloid A (SAA) protein in
chronic inflammation. SAA fragments deposit as amyloid fibrils
predominantly in the kidney. The primary strategy for treatment has been
controlling the inflammation though sometimes the underlying cause might
remain unidentified. Plasma SAA concentration kept below 10mg/L correlates
with disease activity and is a prognostic marker [59]. Antisense
oligonucleotides have been studied pre-clinically in a murine AA model to
suppress serum amyloid A and thus reduce amyloid deposition [60]. Peak
SAA levels in the mice were reduced as much as 65% compared to control.
Sandy W. Wong and Raymond L. Comenzo
36
TARGETING THE DEPOSITS AND FIBRILS
WITH IMMUNOTHERAPY
Serum amyloid P component (SAP) is a normal circulating protein also
known as pentraxin-2. In systemic amyloidosis, SAP is universally deposited
on amyloid fibrils. SAP promotes amyloid fibrillogenesis in vitro [61] and
prevents proteolysis of amyloid fibrils of AL and AA amyloidosis [62].
Human neutrophil cathepsin G and elastase digestion of amyloid fibrils in
vitro was blocked by SAP, only when SAP was bound to the fibrils [62].
Circulating SAP thus does not offer a protective effect to the amyloid fibrils
against proteolysis. Furthermore, amyloid deposition in AA amyloid murine
model was delayed in mice with deletion of the SAP gene [63]. Thus,
depletion of SAP may render amyloid fibrils susceptible to proteolysis and
reduce fibrillogenesis. In addition, the presence of SAP on the amyloid fibrils
provides a universal amyloid target for monoclonal antibody-mediated
clearance.
During the investigations into SAP’s protective effects on amyloid fibril
proteolysis, a cyclic pyruvate acetal of galactose, methyl 4,6-O-(1-
carboxyethylidene)-b-D-galactopyranoside (MOβDG) was found to inhibit
binding of SAP to amyloid fibrils and could completely dissociate SAP from
amyloid deposits in vitro [62, 64-66]. As such, a compound was identified
during a high throughput screen for inhibitors of SAP binding to Aβ amyloid
fibril binding. R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]
pyrrolidine-2-carboxylic acid (CPHPC) was identified as a competitive
inhibitor of SAP binding to amyloid fibrils [66]. Unexpectedly CPHPC, a
palindromic compound, crosslinked 2 SAP pentamers, leading to rapid
clearance by the liver producing not only a profound depletion of circulating
SAP but also removal of SAP from human amyloid tissue tissues in a clinical
trial [66]. Seven patients with amyloidosis (6 AL, 1 AA, 1 localized AL) were
given CPHPC by intravenous infusion. Interestingly, plasma SAP normalized
by 48 hours for patients with little or no amyloid but patients with a significant
amyloid burden had low serum SAP for a prolonged period of time, as long as
20 days in one patient [66]. By
123
I-labeled SAP scintigraphy, patients who
received CPHPC administration showed marked accumulation of tracer in the
liver consistent with liver uptake of SAP [66].
A second phase I clinical trial with CPHPC enrolled 31 sequential patients
with AL, hereditary fibrinogen amyloidosis (Afib), and ATTRmt [67]. CPHPC
produced sustained >95% depletion of circulating SAP in all patients and 90%