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Figure 5. In vivo transduction with NHP preinjection serum-incubated AAV mix. (a) Schematic of transduction of engrafted FRG mice with coincubated AAV
mix and antibodies from the postapheresis/preinjection step of the experiment using Macaca fascicularis NHP. (b) NGS read contribution (%) for each AAV
from extracted xenograft DNA in absence or presence of serum in indicated dilutions. Cells were sorted for xenograft species. (c) NGS read contribution (%)
for each AAV from mouse extracted DNA in absence or presence of serum in indicated dilutions. Cells were sorted for mouse origin. All data shown as ''Rh/Cy/
hFRG no sera'' are also used for Fig. 2b and are shown again for ease of comparability. hFRG, humanized FRG.
study in 85 human samples. Samples were collected from
individuals younger than 1-year old (17%), aged 1-5 years
(35%), 6-10 years (16%), 11-20 years (12%), and older
than 20 years (20%). The overall seroprevalence of NAb
ranged from 14% (AAV5) to 29% (AAV3b) (Fig. 6a).
Seroprevalence was low during the first years ofage and
increased after the age of 10 years (Fig. 6b). NAb titers are
higher for AAV3b, AAV2, andAAV-NP59 with median of
1/320, 1/160 and 1/80, respectively. Lower NAb titers were
observed for AAV5, AAV8, and AAV-LK03 with medians
of 1/5, 1/5 and 1/20, respectively (Fig. 6c). Strong crossreactivity
with other liver-tropic capsids tested was detected
for AAV2, AAV-NP59, and AAV5 (Fig. 6d).
In the cohort of plasma samples tested for seroprevalence,
80% were from persons aged 20 years or
younger. This work corroborates previous findings that
most pediatric individuals have not yet developed antiAAV
antibodies, which emphasizes a decisive immunological
advantage of targeting this age group.63,64 The
seroprevalence rates increased rapidly in teenage and
adulthood where NAb seroprevalence rates could be as
high as 80% for the some AAV serotypes.27,64-68 Our data
for sera from the adult population showed lower neutralization
rates compared with other seroprevalence studies66
but the findings were in accordance with our previous
work in the British population.46 AAV2, AAV3b, and
surprisingly AAV-NP59 showed higher titers compared
with AAV5, AAV8, and AAV-LK03, although the use of a
higher dose for AAV5 and AAV8 may have reduced the
sensitivity and partly underestimated the titers.69
NAb cross-reactivity was high between some livertropic
capsids.46,64,66 These findings support the need for
innovative immunosuppression protocols or antibody reduction
methods to allow successful transduction in preimmunized
patients and readministration, if needed.62
Antibody reduction has previously been performed either
by removing all antibodies (as has been used for the NHP
in the presented study)47 or by specifically removing antiAAV
antibodies.70,71 Another recently reported option is
to use IgG degrading enzymes, which would be infused
systemically before AAV delivery.72,73 These methods
would be especially relevant for use in infants, who are
only passively immunized through adoptive transfer from
the mother and do not have memory cells specific against
AAV epitopes, making it less likely for their immune
system to be activated.

Human Gene Therapy - April 2023

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