Improved Brain Expression of Anti-Amyloid β scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer
Authors: Perche Federico , Uchida Satoshi , Akiba Hiroki , Lin Chin-Yu , Ikegami Masaru , Dirisala Anjaneyulu , Nakashima Toshihiro , Itaka Keiji , Tsumoto Kohei , Kataoka Kazunori 
Published: December 2016

Background: The ever-increasing number of people living with Alzheimer’s disease urges to develop
more effective therapies. Despite considerable success, anti-Alzheimer immunotherapy still faces the
challenge of intracerebral and intracellular delivery. This work introduces in situ production of anti-amyloid
beta (A&#946;) antibody after intracerebral injection of PEG-PAsp(DET)/mRNA polyplexes as a novel immunotherapy
approach and a safer alternative compared to high systemic antibodies doses or administration of
adenovirus encoding anti- A&#946; antibodies.

Methods: We used mRNA encoding three different A&#946;-specific scFV with a secretion signal for passive
immunotherapy. scFv contained a 6xHis-tag for immuno-detection. The secretion signal from IL2 (IL2ss)
was added to allow extracellular engagement of senile plaques. A&#946; affinity of scFv was measured by surface
plasmon resonance. To allow intracellular delivery, scFv were administered as polyplexes formed with our
smart copolymer polyethylene glycol-poly[N’-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PEG-PAsp
(DET)]. We evaluated scFv expression in cellulo by Western blot and ELISA, their ability to disaggregate
amyloid aggregates by thioflavine T assay. Moreover, in vivo expression and therapeutic activity were evaluated
in a murine amyloidosis model, by anti-6xHis-tag ELISA and anti- A&#946; ELISA, respectively.

Results: The selected anti-amyloid beta scFv showed affinity towards A&#946; and disaggregated A&#946; fibers in
vitro. Whereas both DNA and mRNA transfection led to scFV expression in cancer cells, only mRNA led to
detectable scFv expression in primary neurons. In addition, the use of IL2ss increased by 3.4-fold scFv secretion
by primary neurons over mRNA polyplexes devoid of secretion signal. In vivo, a 3 to 11- fold of intracranial
scFv levels was measured for mRNA compared to DNA polyplexes and higher in vivo scFv levels were
obtained with mRNA containing IL2ss over non-secreted mRNA. Intracranial injection of anti-A&#946; mRNA
polyplexes with IL2ss resulted in 40 % A&#946; decrease in an acute amyloidosis model; with no decrease detected
with control scFv mRNA nor DNA polyplexes. However, no A&#946; decrease was detected in a more challenging
transgenic model of Alzheimer’s disease.

Conclusion: Our results introduce a concerted approach not only for Alzheimer’s disease treatment but also
for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial
delivery of anti-A&#946; scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement
of A&#946; in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary
neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease
in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer,
IL2ss) were integrated into a single formulation.