Supplemental material – Preclinical AAV2.5-Minidystrophin Studies
A total of three late preclinical studies were conduct to examine AAV2.5-Minidystrophin toxicity
and biodistribution, including interaction effects with standard pharmacological treatment of DMD.
Two separate animal studies were conducted to examine any potential adverse interactions
between prednisone and AAV. Study 1 was conducted at the Ohio State University & University
of Florida National Gene Vector Laboratory Toxicology Center, and Study 2 was conducted at The
Ohio State University and Bioreliance:Invitrogen. A final comprehensive study of toxicity and
biodistribution was conducted by Bioreliance:Invitrogen. These three studies are summarized
below.
Prednisone AAV-Minidystrophin Co-delivery in mdx Study 1
Transgenic mdx mice received i.m. vector (1 x 10E11 vg/kg) plus a bolus of prednisone, vector
plus chronic prednisone, or vehicle control (n=3 per group). Animals were sacrificed at 6 weeks
post vector administration. For the purpose of histopathological analysis, tissue from the brain,
lung, gonad, heart, spleen, kidney liver and injected and un-injected muscle were examined.
Vector DNA was detected in injected muscle samples from all animals receiving vector (at >100
copies of single stranded vector DNA/ug genomic DNA), and not from animals receiving vehicle.
This study concluded that treatment of male mdx mice with vector and bolus Prednisone or vector
and chronic Prednisone, did not induce histologic treatment-related effects.
Prednisone AAV-Minidystrophin Co-delivery in mdx Study 2
Transgenic mdx mice received intramuscular vector (1x10E11vg/kg), vector plus a bolus of
prednisone, or vector plus chronic prednisone to mimic the proposed protocol (n=6 per treatment).
We examined vector genome copies in the injected and contralateral control muscle, as well as
conducting histopathological examination of the injected and contralateral control muscle. Animals
were sacrificed at 6 weeks or 12 weeks post vector administration (n=3 per group). There were no
significant differences in dystrophin expression between the group receiving vector plus bolus
Prednisone or the group receiving vector only at 6 and 12 weeks. Both groups showed a maximum
of dystrophin expression of 30% in the treated muscle at both time-points. Animals treated with
vector and chronic Prednisone showed a maximum dystrophin expression level ~10% higher
(40%) than the vector only animals at 6 and 12 weeks. Vector genomes were present only in the
injected muscles (>100 copies/ug genomic DNA). There was no evidence of toxicity in the vector
treated muscles. No treatment related abnormalities were found in the liver, testes, or heart at
either time-point.
Comprehensive Thirty-Six Week Toxicity and Biodistribution Study in Naïve Mice
The purpose of this study was to assess the potential toxicity, biodistribution and elimination of an
Adeno Associated Virus Vector BNP2.5 CMV-3978 (AAV2.5.CMV.mini-dystrophin viral
genome) following a single intramuscular injection in mice. All portions of the study performed by
BioReliance were in compliance with Food and Drug Administration Good Laboratory Practices
as found in title 21 Code of Federal Regulations part 58 and the Organization for Economic
Cooperation and Development (OECD) Principles of Good Laboratory Practice, revised 1997
(ENV/MC/CHEM (C(97)/186/Final). The number of animals, animal procedures and experimental
design for this study were reviewed and approved by the BioReliance Institutional Animal Care
and Use Committee #220.
This report presents the results of a thirty-six week toxicity and biodistribution study. In this study,
two groups of 27 male C57BL/I0 mice (including three extras) were treated with a single
intramuscular injection of the formulated test article (BNP2.5 CMV-3978) at 1 x 1011 and 1 x 1012
vector genome/kg in a 20μl dose volume (Groups 2 and 3, respectively). A control group,
consisting of 27 male mice (including three extras) was treated in the same manner as test article
treated groups but received only the vehicle (Phosphate Buffered Saline with 5% sorbitol). The test
material or vehicle was administered by intramuscular injection into the center of the tibialis
anterior muscle of the right hind limb on Day 1. Eight animals/group were sacrificed 6 and 12
weeks post injection (5 for toxicology, histopathology and biodistribution evaluation and 3 for
antibody and gene expression evaluation). Tissues collected during necropsy at the 36-week time
point were preserved in 10% buffered neutral formalin and those from the control and test article
treated dose groups were embedded in paraffin and archived at BioReliance without further
processing or examination. Week-36 tissues for biodistribution analysis were collected, frozen and
archived at BioReliance without analysis. Week-36 blood for biodistribution analysis was
collected, DNA extracted and the serum harvested. These samples were frozen and archived at
BioReliance without analysis. Blood serum and skeletal muscle for anti-dystrophin antibody
analysis was collected, frozen and archived at BioReliance without further analysis. All specimens
will be held at BioReliance archives for the minimum retention period specified by the regulatory
agencies. The three extra animals were assigned to each group to serve as back-ups in case of
losses during the study.
All animals were monitored within approximately two hours after injection on Day 1 and followup observations were performed at 4 and 8 hours post-injection and daily thereafter. At 24 hours
post-injection (and on the day of sacrifice) each mouse was individually observed in a separate
holding cage and graded for skin color, spontaneous activity, gait and tail elevation during forward
motion. Observations for moribundity and mortality were performed twice daily for signs of illness
or toxicity. A detailed (hands-on) examination was performed on all animals on Day 1 and weekly
thereafter at the time the animals were weighed. Body weights were measured on Day 1 (prior to
dosing); 24 hours post injection, weekly for 12 weeks and every other week thereafter. Body
weights were also collected prior to fasting before scheduled sacrifice.
Twenty-four hours post injection, blood from all animals was collected for PCR analysis, but per
protocol amendment the analysis was not performed. Five animals scheduled to be sacrificed at 6
weeks and 12 weeks post injection were bled and serum and whole blood samples were shipped to
a subcontractor clinical pathology laboratory for evaluation. Up to 5 animals per group designated
for the Toxicity/Biodistribution Study and up to 3 animals per group designated for the antidystrophin antibody and gene expression study components were sacrificed 6 and 12 weeks post
injection and necropsied. At sacrifice, the animals were weighed to the nearest 0.1 gram. Animals
designated for the Toxicity/Biodistribution Study were anesthetized with 70% CO2/30% O2, in
sequence of intended necropsy, and whole blood and frozen serum was collected and stored at
42°C or -60°C, respectively. The whole blood samples were shipped to the PCR laboratory and
the frozen serum samples, from animals sacrificed at 6 and 12 weeks only, were sent to Dr. Xiao
Xiao for anti-AAV antibody evaluation.
Following blood collection from the animals sacrificed at 6 and 12 weeks, the animals designated
for the Toxicity/Biodistribution Study were euthanized by CO2 overdose and selected tissues
collected. Vehicle-treated animals were sacrificed prior to vector-treated animals. A complete PCR
necropsy was conducted and abnormalities recorded. Additionally, selected tissues were collected
from 3 separate study animals surviving to scheduled sacrifice and sent to the University of
Pittsburgh for anti-dystrophin antibody analysis. Frozen serum from the 3 separate study animals
was also sent to the Dr Xiao Xiao for anti-dystrophin antibody evaluation.
An extensive conventional necropsy was performed following the PCR necropsy for animals
designated for the Toxicity Study. Gross observations noted during necropsy were recorded.
Selected tissues/organs and all gross lesions were collected and fixed in 10% neutral buffered
formalin. Tissues from the high-dose and vehicle control groups were embedded in paraffin,
sectioned at 6 microns or less, stained with hemotoxylin and eosin. For the animals sacrificed at 36
weeks, tissues were also processed in the mid-dose group. A microscopic examination was
performed on animals sacrificed at 6 and 12 weeks, but the samples were not examined, only
archived, for the animals sacrificed at 36 weeks.
Treatment with the test article did not affect survival, body weights or total body weight gain. Shut
eyes and/or eye opacity was observed in two control animals, fifteen Group 2 animals and
seventeen Group 3 animals. There appeared to be a decrease in BUN for Groups 2 and 3 in weeks
6 and 12, but this was due to the control being high at these time points. The control had
normalized by week 36 and the treated animals had similar values throughout the study so none of
these findings are considered significant. There were no test article-related effects on clinical
hematology parameters or gross necropsy observations. Two of the five animals examined
microscopically at the 6-week time point exhibited either a mild or moderate degree of subacute
inflammatory foci within the interstitium of the SOI skeletal muscles. However, there were no
treatment-related significant lesions observed in the 12 week study animals. Week 36 tissue
samples were archived at BioReliance without examination. PCR biodistribution evaluation
indicated that the vector was concentrated in the injected muscle tissue at both the 6 and 12 week
time points. PCR evaluation of blood samples was non-informative. A reanalysis of eight Week 6
blood samples (four vehicle control and four high dose) was performed. All vehicle samples were
negative and all high-dose samples were positive. Week 36 samples were archived at BioReliance
without analysis.
Overall, BNP2.5 CMV-3978 vector caused no significant toxicity in C57BL/10 mice. All animals
survived until scheduled sacrifice with the exception of one extra group 3 animal, which was
removed on Day 155 due to lack of documentation of dosing. PCR biodistribution evaluation
indicated localization primarily at the site of muscle injection which persisted for at least 12 weeks.