Gelsolin amyloidosis (AGel), formerly known as familial amyloidosis Finnish type (FAF), is a
debilitating, autosomal, dominantly heritable, incurable disease caused by a point mutation -
most commonly G654A/T - in the GSN gene. As a result, gelsolin’s second domain loses part of
its intrinsic stability and can adopt an intermediate state during Ca2+ activation. This aberrant
conformation exposes an otherwise buried furin cleavage site. During secretion, mutant plasma
gelsolin encounters furin in the trans-Golgi network. Proteolysis produces a C-terminal, 68 kDa
fragment - C68 - which, upon secretion in the extracellular matrix, gets cleaved by MT1-MMPlike proteases. With this, 8 and 5 kDa amyloidogenic fragments are produces which, in time,
start to polymerize into amyloid fibrils and which eventually form amyloid plaques. Patients,
of whom the majority is found in Finland, experience a triad of ophthalmological, neurological
and dermatological symptoms. At the moment, treatment is purely symptomatic; eye drops,
corneal transplantation and plastic surgery. Therapies involving the inhibition of furin and/or
MT1-MMP have been suggested. However, tampering with these key proteases will almost
surely result in severe unwanted side effects. In our lab we therefore redirected our attention
towards mutant plasma gelsolin. Nanobodies were developed against both gelsolin and the 8 kDa
amyloidogenic fragment. Upon in vitro screening a chaperone for mutant plasma gelsolin - Nb11
- and chaperones for C68 - FAF Nb1, 2 and 3 - (partly) protected their antigen against furin
and MT1-MMP respectively. Intraperitoneal injection with FAF Nb2 and in vivo expression of
Nb11 in the AGel mouse model both resulted in a reduced deposition of the amyloidogenic 8 kDa
fragment. In this thesis we further explored the potential of these nanobodies in regard to AGel
therapeutics.
In a first study the imaging potential of the FAF nanobodies was explored. The rationale for this
was that, up until now, the efficacy of AGel therapeutics under development could only be measured via end-stage animal trials. With the already characterized FAF Nbs - raised against the 8
kDa amyloidogenic gelsolin fragment - we set out to shed light on this black box hurdle. Coupled
to 99mTc, all three nanobodies rendered clear, low background, SPECT/CT images of the gelsolinamyloid buildup in AGel mice. Based on its superior signal-to-noise ratio and signal specificity, FAF Nb1 was chosen as the most promising candidate. A follow-up study involving AGel mice expressing the therapeutically active Nb11 demonstrated that, besides rendering qualitative images, FAF Nb1 can also provide quantitative data. Indeed, comparison of quantified SPECT/CT
images, biodistribution analysis and immunofluorescent histology revealed similar patterns in signal increase. Furthermore, a correlation plot showed a positive relationship between 99mTc-FAF
Nb1 uptake and in vivo gelsolin amyloid buildup.
In a second study the two types of therapeutic chaperone nanobodies - Nb11 shields mutant
plasma gelsolin form furin and FAF Nb1, 2 and 3 shield C68 from MT1-MMP - were combined
into a single bispecific format. The FAF Nbs were linked head-to-tail to Nb11 via a MT1-MMP
sensitive linker. We hypothesized that during secretion, the bispecific Nb would encounter mutant
plasma gelsolin in the trans-Golgi network and (partly) shield it from furin degradation. Upon
secretion in the extracellular matrix, the MT1-MMP sensitive linker would serve as a decoy and,
at the same time, release the FAF Nb moiety, which would then shield (the lower amount of)
C68 from MT1-MMP degradation. Overall this double hit strategy would result in a significant
decrease in amyloidogenic 8 kDa peptide production. In vitro, a combination of Nb11 and
FAF Nb1 proved to be the most potent. As this approach requires intracellular delivery of the
protective bispecific nanobody, adeno-associated virus serotype 9 (AAV9) gene therapy seemed
an appropriate and clinically relevant method of administration during an intervention study in
AGel mice. FAF Nb1 based SPECT/CT imaging, and immunohistochemistry evinced a significant
decrease in gelsolin amyloid buildup, which translated in improved muscle contractile properties.