499 Development of AAV2/8 Mediated Gene Therapy Clinical Trial for Crigler Najjar Syndrome Type I Optimization of Liver Specific Expression Cassette Molecular Therapy Volume 21, Supplement 1, May 2013[.]
Trang 1Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
S192
GENE & CELL THERAPY OF DIABETES, METABOLIC AND GENETIC DISEASES II
497 Prevention of Neuropathology and Normal
Cognitive Development in the Hyperargininemic
Mouse with AAV Gene Transfer
Ellen K Lee,1 Chuhong Hu,1 Ragini Bhargava,1 Ravi Ponnusamy,2
Hana Park,1 Sarah Novicoff,1 Nora Rozengurt,3 Bart Marescau,4
Peter De Deyn,4 David Stout,5 Lisa Schlichting,6 Wayne W Grody,7
Stephen D Cederbaum,7 Gerald S Lipshutz.1
1 Surgery, David Geffen School of Medicine at UCLA, Los Angeles,
CA; 2 Psychology, David Geffen School of Medicine at UCLA, Los
Angeles, CA; 3 Pathology and Laboratory Medicine, David Geffen
School of Medicine at UCLA, Los Angeles, CA; 4 Laboratory
of Neurochemistry and Behavior, University of Antwerp and
Institute Born Bunge, Antwerp, Belgium; 5 Molecular and Medical
Pharmacology, David Geffen School of Medicine at UCLA, Los
Angeles, CA; 6 Biochemical Genetics Laboratory, University
of Colorado, Denver, CO; 7 Pediatrics, David Geffen School of
Medicine at UCLA, Los Angeles, CA.
Arginase I defi ciency is the least severe urea cycle disorder and
is characterized by hyperargininemia and infrequent episodes of
hyperammonemia Patients suffer from neurological impairment
with cortical and pyramidal tract deterioration, spasticity, loss of
ambulation, and seizures, and is associated with intellectual disability
In mice, onset is heralded by weight loss beginning around day
15; gait instability follows progressing to inability to stand and
development of tail tremor with seizure-like activity and death We
developed a serotype rh10 adeno-associated virus expressing arginase
under either 1) the CBA promoter or 2) a liver specifi c promoter
(LSP) and followed animals long-term for the development of any
evidence consistent with brain dysfunction Survival was greater
at 6 months in mice injected with the CBA promoter compared to
the LSP promoter With the CBA promoter, arginase is detected in
extrahepatic tissues; however it is unclear if functional protein can
be produced at these sites An extensive battery of behavioral testing
was performed on the AAV-CBA mice including SHIRPA, open
fi eld, elevated plus and Morris water maze, hot plate pain assay, and
rotarod and demonstrated no differences from controls Furthermore,
histopathological evaluation demonstrated that treated mice were
indistinguishable from littermates and that putative compounds
associated with neurotoxicity (guanidino compounds; GCs) were
diminished but not normal later in life In addition, treatment resulted
in near complete resolution of amino acid and ammonia levels
early in life; however there was development of some metabolic
derangement later with decline in transgene expression, more so in
the LSP mice Ammonium challenging at 4 months revealed that
treated mice were affected by exogenous loading much greater than
littermates These results demonstrate that AAV-based therapy for
hyperargininemia can be effective and prevent the development of
neurological abnormalities and cognitive dysfunction in a mouse
model of hyperargininemia As GCs do become elevated later in life
with no obvious resultant CNS abnormality, these data suggest that
the hypothesis that GCs are the cause of the unique neurotoxicity in
hyperargininemia may not be correct Finally, nitrogen challenging
reveals that these gene therapy treated mice remain impaired in the
handling of waste nitrogen and are vulnerable
498 Assessing the Safety of AAV Liver Gene Transfer of Hyperfunctional FIX Padua in Inhibitor-Prone Dogs and Mice
Julie M Crudele,1 Jonathan D Finn,1 Joshua I Siner,1 Yifeng Chen,1 Shangzhen Zhou,1 Glenn P Niemeyer,2 Clinton D Lothrop
Jr,2 Federico Mingozzi,1 Katherine A High,1 Valder R Arruda.1
1 Children’s Hospital of Philadelphia, Philadelphia, PA; 2 University
of Alabama, Birmingham, AL.
Clinical trials have shown that the safety of adeno-associated viral vector (AAV) gene transfer can be limited by dose-dependent immune responses against the capsid Modifi cations to the transgene to increase the specifi c activity of a protein could allow for lower vector doses to be given, provided the neotransgene is not immunogenic Previously we showed that the naturally occurring gain-of-function Factor IX R338L (FIX-Padua) exhibits ˜8-fold increased specifi c activity in humans (N Engl J Med 2009) In hemophilia B (HB) dogs with a missense mutation in the canine (c) F9 gene, delivery of AAV6-cFIX-Padua to skeletal muscle resulted in similar increased specifi c activity with no anti-FIX neutralizing antibodies (inhibitors)
or FIX-specifi c T-cell response (Blood 2012) Here we tested the effi cacy and immunogenicity of cFIX-Padua in a severe HB dog colony with an early stop codon mutation prone to develop FIX inhibitors A naive dog treated with a liver-specifi c AAV8-cFIX-Padua showed expression levels of ˜2% antigen and ˜23% activity, with no inhibitor development Whole blood clotting time (WBCT) returned
to normal by day 3 post-vector administration and remains stable for >12 months An additional dog had previously being exposed to human (h) FIX protein and developed inhibitors that cross-reacted with cFIX When treated with AAV, anti-cFIX antibodies peaked at day 14 post-AAV, but dropped to undetectable levels by day 70 with
a concurrent rise in expression levels to activity levels of ˜30% and normalization of WBCT We also compared the long-term safety and effi cacy of hFIX-Padua to hFIX-WT in WT mice Five dose cohorts of AAV8-hFIX-WT (2.5x108–5x1010 vg/kg; n=5/group) led to expression ranging from <40% to >600% of normal when measured by antigen
or activity, with a linear correlation of activity/antigen In contrast, AAV8-hFIX-Padua doses (1x108–1x1010 vg/kg; n=5/group) led to antigen levels ranging from <2% to >200% of normal but activity levels of <40% to >1900%, with an elevated specifi c activity ˜9 Markers of pathological activation of coagulation were assayed 2-8 months post-AAV, with no signifi cant differences seen in thrombin-antithrombin complexes or D-dimer as a function of expression of FIX-Padua vs FIX-WT In addition, Kaplan-Meyer survival curves were similar between the groups Finally, mice receiving AAV8-hFIX-WT were immunologically challenged 10-15 weeks after gene delivery with recombinant hFIX-Padua protein, and visa versa, alone
or with CFA In no instance did mice develop antibodies to FIX Together these data show that mice expressing FIX-Padua exhibit
no increased risk of thrombophilia or mortality at the levels studied (up to nearly 2000% activity) compared to FIX-WT FIX-Padua also shows no increase in immunogenicity compared to FIX-WT and is capable of tolerizing HB dogs and mice Thus, FIX-Padua is
an attractive transgene that will allow for decreased vector doses in human HB gene therapy
499 Development of AAV2/8-Mediated Gene Therapy Clinical Trial for Crigler-Najjar Syndrome Type I: Optimization of Liver-Specifi c Expression Cassette
Nunzia Pastore,1 Alberto Auricchio,1,2 Nicola Brunetti-Pierri.1,2
1 Telethon Institute of Genetics and Medicine, Naples, Italy;
2 Department of Pediatrics, Federico II University, Naples, Italy.
Crigler-Najjar syndrome type I is a severe inborn error of bilirubin metabolism due to mutations of the liver-specifi c uridine
Trang 2diphospho-Molecular Therapy Volume 21, Supplement 1, May 2013
CANCER - IMMUNOTHERAPY II
glucuronosyl transferase 1A1 (UGT1A1) gene Affected patients
have increased serum bilirubin that is treated with daily phototherapy
for several hours to prevent irreversible brain damage Despite
treatment, patients remain at risk of life-threatening elevations of
serum bilirubin and often undergo orthotopic liver transplantation
Therefore, alternative therapies are needed to overcome the mortality
and morbidity associated with transplantation procedure, and risks of
life-long immunosuppression AAV2/8 vectors are very attractive for
liver-directed gene therapy and have recently resulted in encouraging
results in a human clinical trial for hemophilia B Gene therapy has the
potential to provide a defi nitive cure for patient with Crigler-Najjar
syndrome type I and our studies focused on the design and optimization
of an AAV2/8 vector for clinical trial Multiple expression cassettes
expressing the UGT1A1 gene inserted into the AAV2/8 vectors were
generated for in vivo investigation in the Gunn rats, the animal model
for Crigler-Najjar syndrome The comparison of the expression
cassettes was performed injecting AAV2/8 vectors into the tail vein
of 4-6 week-old female Gunn rats We fi rst injected animals with a
vector expressing the human UGT1A1 under the control of the liver
specifi c thyroxine-binding globulin (TBG) promoter but only the
high dose of 5x1013 genome copies (gc)/kg resulted in a signifi cant
60% reduction of serum bilirubin levels by 12 weeks post-injection
Next, we achieved a 50% reduction of baseline serum bilirubin with
5x1012 gc/kg of a vector encoding the human UGT1A1 under the
control of the liver-specifi c LP1 regulatory element that consists of the
core domains from the human apolipoprotein hepatic control region
and the human alpha-1-antitrypsin gene promoter Interestingly, the
injection of the same dose of another vector bearing codon optimized
human UGT1A1 cDNA under the control of the same LP1 promoter
resulted in complete normalization of serum bilirubin levels by 2
weeks post-injection and the correction was sustained for all the
time of the observation In summary, our study shows that AAV2/8
vector with codon optimized UGT1A1 gene under the control of the
hepatocyte-specifi c LP1 promoter results in improved and sustained
correction of hyperbilirubinemia in Gunn rats Taken together, these
data demonstrate the development of an optimal expression cassette
for liver-directed gene therapy of Crigler-Najjar syndrome type I and
form the preclinical basis for the development of a gene therapy trial
for this severe disorder
500 Stromal Cell-Derived Factor-1 Non-Viral
DNA Therapy Accelerates Wound Healing and
Decreases Scar Formation in Porcine Surgical
Incisions
Timothy J Miller,1 Karissa Henson,1 Rahul Aras,1 Evan Facher,1
Marc S Penn.2,3
1 SironRX Therapeutics, Inc, Cleveland, OH; 2 Summa Health
System, Akron, OH; 3 Northeast Ohio Medical University,
Rootstown, OH.
JVS-100 is a non-viral gene therapy that expresses human Stromal
cell-Derived Factor-1, (SDF-1, aka CXCL12) in target tissue SDF-1
is a strong chemoattractant of bone marrow derived stem cells and
tissue specifi c progenitor cells to the site of tissue damage, which
promote tissue preservation and blood vessel development SDF-1
triggers a number of protective molecular cascades that are both
anti-infl ammatory and anti-apoptotic Endogenous SDF-1 expression
is increased in damaged tissue and has been demonstrated to be a
pro-healing chemokine, but expression lasts for less than a week,
and therefore the induced stem cell homing response quickly fades
SDF-1 gene transfer to prolong or re-introduce SDF-1 expression at
the site of tissue damage has been proposed as a potential therapeutic
strategy for treatment of wound healing based on animal studies
by several independent laboratories We demonstrate that
non-viral gene therapy with JVS-100 to increase the duration of SDF-1
expression in the skin around wounds is a safe and effi cacious
means to accelerate wound repair and decrease scar formation in clinically relevant porcine wounds Full thickness incisions were created on the backs of juvenile female Yorkshire pigs and JVS-100 was delivered as multiple needle-free subcutaneous injections at the time of wound closure High levels of protein expression were detected at specifi cally at injections sites 3 days post-injection Wound healing was signifi cantly increased 40-120% in all dose groups, with dose-dependent acceleration of wound closure observed at Day 4 (P<0.01), Day 7 (P<0.05) and 14 post-injection (P<0.05) Visible scar formation was reduced 20-100% compared to no treatment or vehicle treated controls at 28 (P<0.02) days post-injection compared to no treatment controls Scars were evaluated using the Manchester Scar scale and demonstrated improvement as early as Day 7 Scar color was signifi cantly reduced Day 28 days after treatment (P<0.005) in JVS-100 injected wounds compared to no treatment Additionally, wounds treated with JVS-100 demonstrated that JVS-100 restored wounded skin strength and durability similar to unwounded skin Treated wounds regained approximately 60% of the tensile strength of unwounded skin compared to 30% of no treatment or vehicle treated wounds (P<0.05) Blood vessel density was increased 35% over controls 60 days post-treatment at the highest doses tested (P<0.05) This data was the basis for a randomized, double-blind, placebo controlled, dose escalation clinical trial that is currently enrolling subjects and evaluating safety and preliminary effi cacy of JVS-100
in the treatment of subjects with a high body mass index (BMI greater than 25) receiving sternotomy wounds during cardiothoracic surgery
Cancer - Immunotherapy II
501 DAI-Armed Double Deleted Oncolytic Vaccinia Virus Displays Enhanced Anti-Tumor Activity by Eliciting a More Robust Anti-Tumor Immune Response
Mari Hirvinen,1 Kilian Guse,2 Marko Ahonen,2 Eerika Karli,2 Dario Greco,3 Markus Vähä-Koskela,2 Akseli Hemminki,2 Vincenzo Cerullo.1
1 Laboratory of Immunovirotherapy, Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland; 2 Cancer Gene Therapy Group, Department of Pathology and Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland;
3 Finnish Institute of Occupational Heath, Helsinki, Finland.
Oncolytic viruses are an attractive platform for cancer therapy and immunotherapy Viral replication induces cell death with subsequent release of tumor-associated antigens (TAAs) that can be cross-presented on MHC-I by professional antigen presenting cells such as dendritic cells This can lead to activation and expansion of tumor-specifi c T cells that can reach metastasis and develop into a subpopulation of long-lasting memory T cells Oncolytic viruses have the ability to enhance the TAA release by oncolysis and simultaneously provide DCs with the necessary co-stimulatory signals or “danger signals” to promote cross-priming of nạve T cells This is also linked
to their ability to interact with pattern recognition receptors (PRRs) DAI (DNA-dependent activator of IFN-regulatory factors), also known as Z-DNA binding protein 1 (ZBP1), is a potent activator of the immune response In this work, we have fi rst identifi ed DAI as a new PRR involved in triggering a type I IFN-mediated response following the double deleted oncolytic vaccinia virus (VVDD) infection We showed that type I IFNs and pro-infl ammatory cytokines secretion was signifi cantly reduced in DAI-silenced Jurkat cells following the infection with the virus Despite its astonishing performance in animal models and clinical trials, the capacity of vaccinia virus to trigger a tumor-specifc immune response has been disappointing thus far To improve that, we hypothesized that arming VVDD with DAI would enhance the vaccine properties of this virus To this end