ChasRiver_Aug2022_Perspectives-Toward-a-Treatment - 9

PERSPECTIVES
but had already done much of the legwork to develop a
more stable plasmid that has shown higher fidelity within
those ITR regions. Working with Reno's team, we were
able to insert our gene-of-interest into that plasmid. After
production, we found those ITRs were intact, normal,
and faithful to our sequence. That's a nice example of
overcoming an anticipated problem leveraging Charles
River's scientific expertise.
RENO FRANCHETTI:
To recognize this problem from the start, you must have a
robust plasmid screening process. We always run a full NGS
(Next Generation Sequencing) on both the seed and the first
amplified plasmid. With NGS that can read through ITR
regions you can recognize alterations, deletions, or mutations.
Upon discovering these changes, we wanted to communicate
this promptly. We discovered this at an early stage, so
it did not slow down the program. In parallel to scaling this
process up to 50 L, we were able to test Paros Bio's expression
cassette in three different plasmid backbones that our
scientists have developed.
We screened them again for productivity and stability
through restriction enzyme digest and additional NGS to
confirm the stability of these ITRs within these plasmid
backbones. We took all three new constructs and did a smallscale
packaging of AAV to ensure that packaging did not lose
any efficiency. Meanwhile, we were still scaling this process
up to 50 L. Then we were able to take the new chosen
plasmid construct and drop it into the original scope of work
to scale its manufacturing. It resulted in good productivity
and stability of the packaging at the 50 L scale.
Anjali Sarkar: What kinds of models and
studies are you using to test safety and
efficacy of the PSEN1 gene therapy?
HOLDEN JANSSENS:
On the in vivo side, our work looked into determining the
efficacy and distribution of the AAV-being able to administer
the test materials into mice in different brain regions, to
determine where the best locations for those injections were,
in terms of biodistribution of the AAV throughout the brain,
how many injections were needed, and the titer. We looked at
a variety of different AAVs to determine the optimal one. We
explored which method had optimal biodistribution as well as
other downstream readouts, starting with a standard wildtype
animal and then moving into a transgenic model of AD.
BEN MOORE:
Our approach with in vivo models has been to escalate
toward translatability. We began basic work in wild type
animals to understand how our virus spreads and expresses
our gene-of-interest. Working closely with Holden's team, we
were able to optimize areas of the brain we could inject into
to achieve broad and robust coverage.
We then leveraged this model development, escalating to a
model that functionally lacked PSEN1. In this model we were
able to understand target engagement, how our gene incorporates
into a functional γ-secretase complex and how that
incorporation improves efficacy, function, and even rescues
neurodegeneration. From there, we escalated to a mouse
model that is as close as we can currently get to the human
disease. Using a model that carries a copy of the human
mutant PSEN1 we established that our therapeutic achieves
efficacy and normalizes γ-secretase function to wildtype
levels, even in the presence of the loss-of-function mutation.
We've worked closely with Holden's group to run much of
our in vivo program here at Paros Bio. We have done several
surgical projects on different mouse models collaborating
with their group, where they have been directly injecting our
therapeutic or other vectors into mouse brains. Overall, we've
been incredibly happy with this collaboration, specifically
with the consistency of this challenging surgical technique.
After the surgery, in general, they house the mice for us for a
few months and then we're able to collect tissue from those
animals for our internal characterization. This relationship
has been a major driver of our preclinical efficacy/proof-ofconcept
program. We've worked with them from the early
GENengnews.com | 9
https://www.criver.com/drug-discovery-to-first-in-human http://www.GENengnews.com

ChasRiver_Aug2022_Perspectives-Toward-a-Treatment

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