547 Concentration of AAV8 and 9 Vector to Titers Ranging from 1 6 x1014 vg/mL Feasibility Assessment for Volume Limited Dosing Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The Ameri[.]
Trang 1Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
S210
AAV VECTORS III
in the heart of Balb/c mice However, detailed protein and mRNA
data demonstrated that these promoters also showed expression in
the liver To further restrict transgene expression, with the goal of
eliminating liver expression, we employed a microRNA-binding site
from mir122 This new data demonstrates that the transcriptional and
post-transcriptional manipulation facilitates cardiac targeting Three
copies of the binding site for liver specifi c mir122 were inserted into
the 3’UTR of AAV9-CMV-EF1a-Luc and injected into Balb/c mice
Bioluminescence imaging showed decreased luciferase signal in the
upper abdominal region and after homogenization liver tissue revealed
a 30 folds decrease in luciferase activity versus the same gene
without mir122 binding sites When mir122 were used in the
AAV9-aCARD-EF1a-Luc, exclusive heart luciferase expression and none
liver expression were observed in Balb/c mice with bioluminescent
imaging and in vitro luciferase assay, while genome copy number
ratio remained unchanged The same result was confi rmed in S100A1
knock-out mice with C57/B6 background To apply this targeting
strategy in gene therapy of heart failure, mir122 binding sites were
inserted into the 3’UTR of AAV9-aCARD-EF1a-S100A1, and
therapeutic effect of S100A1 will be studied in S100A1 knock-out
mice post systematic delivery in a myocardial infarct model In
conclusion, we have successfully restricted transgene expression
to heart in both Balb/c and C57/B6 mice by using cardiac biased
promoter aCARD and mir122 binding sites without altering the
tropism of AAV9 This vector is promising for gene therapy of heart
failure via systematic gene delivery
545 Reprogramming Adipose Tissue Derived
Mesenchymal Stem Cells (AT-MSC) into Pluripotent
Stem Cells by a Mutant AAV Vector
Mong-Jen Chen,1 Yuanqing Lu,1 Takashi Hamazaki,1 Hsin-Yin
Tsai,1 Kirsten Erger,1 Thomas Conlon,1 Arun Srivastava,1 Mark
Brantly,1 Vince Chiodo,1 William Hauswirth,1 Naohiro Terada,1
Sihong Song.1
1 Departments of Pharmaceutics, Pathology, Pediatrics and
Molecular Genetics and Microbiology, University of Floida,
Gainesville, FL.
Induced pluripotent stem (iPS) cells have great potential for
personalized regenerative medicine Although several different
methods for generating iPS cells have been reported, improvement
of its safety and effi ciency is imperative In this study, we tested the
feasibility of using recently established triple tyrosine-mutant AAV2
(Y444+500+730F) vector, designated as AAV 2.3m, to generate iPS
cells We developed a polycistronic rAAV2.3m vector expressing
3 reprogramming factors, Klf4, Oct4, and Sox2, and then used this
vector to infect mouse adipose derived mesenchymal stem cell
(AT-MSC) to induce the generation of iPS cells We demonstrated that
(i) triple tyrosine mutant AAV2 vector is able to reprogram mouse
adult adipose tissue-derived stem cells into pluripotent state Those
rAAV2.3m derived iPS (rAAV2.3m-iPS) cells express endogenous
pluripotency associated genes including Oct4, Sox2, and
SSEA-1, and form teratomas containing multiple tissues in vivo; (ii)
c-Myc, an oncogene, is dispensable in rAAV2.3m mediated cellular
reprogramming; (iii) transgene expressions are undetectable after
reprogramming, while vector DNA is detectable, indicating transgenes
are silenced; and (iv) rAAV2.3m-iPS cells can be further differentiated
into hepatocyte-like cells in vitro and produce liver specifi c proteins
These results indicated the rAAV vector may have some advantages
in generating iPS cells
546 Optimized Kidney-Targeted Gene Delivery Using AAV in a Mouse Model of Cystinosis
Celine J Rocca,1 Sarah M Ur,1 Corinne Antignac,2 Jude R Samulski,3 Stephanie Cherqui.1
1 Pediatrics, UCSD, La Jolla, CA; 2 INSERM U983, hôpital Necker-Enfants Malades, Paris, France; 3 UNC Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
A wide range of monogenic kidney disorders have been identifi ed and thus far no gene therapy approach has been developed to specifi cally target the kidney, even though renal transplantation is associated with signifi cant morbidity and mortality Moreover, due
to the severe shortage of donor organs, patients may wait three to six years for transplantation The main goal of our project is to develop
an effi cient and minimally invasive kidney-targeted gene delivery system using recombinant Adeno-Associated Viruses (rAAV) As a proof of concept, we are using the mouse model of cystinosis, which develops chronic renal disease similar to the patients Cystinosis is an autosomal recessive metabolic disease characterized by intracellular accumulation of cystine The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin Affected individuals typically present with proximal tubulopathy before one year of age and eventually progress to end-stage renal failure Our goal is to optimize kidney-targeted gene delivery via retrograde renal vein injection by testing several rAAV serotypes that have the potential of transducing
a wide range of renal cells We performed injections of rAAV5, 6, 8 and 9 coding for either the luciferase or the green fl uorescent protein (GFP) Our preliminary results showed that rAAV9 is the most suitable serotype to effi ciently target the kidney Indeed, using the IVIS live imaging system and the luciferase assay, we observed that only rAAV9 allows a transduction of both the cortex and the medulla while the other serotypes transduced primarily the medulla This has been confi rmed by confocal GFP observation and GFP-specifi c RT-qPCR quantifi cation We are also testing the Parathyroid Hormone Receptor (PTHR) promoter as a strategy to improve kidney-specifi c gene delivery Once the optimal conditions are defi ned, we propose
to test this approach based on renal vein injection of rAAV-CTNS as
a minimally invasive procedure for treating the renal dysfunction in cystinosis If successful, this strategy may be used in many monogenic hereditary nephropathies
547 Concentration of AAV8 and 9 Vector to Titers Ranging from 1-6 x1014 vg/mL: Feasibility Assessment for Volume-Limited Dosing
J Fraser Wright,1,2 Bernd Hauck,1 Shang-Zhen Zhou,1 Olga Zelenaia,1 Marina Sumaroka,1 Katherine A High.1,2,3
1 Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA; 2 Depts of Pathology and Hematology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; 3 Howard Hughes Medical Institute, The Children’s Hospital of Philadelphia, Philadelphia, PA.
For gene transfer translational research using recombinant AAV, certain clinical programs may require high doses to be administered
in a relatively small volume Furthermore, toxicology studies required to assess investigational product safety at doses typically 10-fold above the anticipated clinical high dose may require vector preparations at very high titers Aggregation is the primary concern for concentrated AAV vectors It was previously reported that recombinant AAV2 undergoes aggregation in a concentration-dependent manner for titers exceeding 1x1013 vector genomes per milliliter (vg/mL; studies herein performed with empty capsid-free vector preparations) when formulated in physiologic ionic strength buffers Elevated ionic strength was found to prevent aggregation
to titers up to 5x1013 (Mol Ther 12:171, 2005); however slightly elevated ionic strength formulations compatible with direct parenteral
Trang 2Molecular Therapy Volume 21, Supplement 1, May 2013
AAV VECTORS III
injection to sites such as CNS / ocular tissues limit AAV2 vector
titers to approximately 2x1013 In the current study the outcome
following concentration of purifi ed recombinant AAV8 and AAV9
by tangential fl ow fi ltration (TFF), a process step compatible with
clinical scale vector processing, was determined Highly purifi ed AAV
vectors recovered following cesium chloride banding were diafi ltered
into 10mM NaPhos, 180mM NaCl, pH7.3 and then concentrated
to the smallest achievable volume by TFF (100kDa MW cutoff)
The titers of the vector at the following stages: 1) before TFF; 2)
following concentration but prior to 0.2micron fi ltration; and 3) after
fi ltration were measured by qPCR and spectrophotometry ( = 260
and 280nm) (Mol Ther 7:122, 2003) Aggregation was assessed by
visual inspection, dynamic light scattering, and spectrophotometry (
= 320nm) For AAV8 and AAV9 vectors, post concentration titers of
1x1014 and 6x1014, respectively, were achieved, without evidence of
aggregation These were the highest titers achievable, limited by the
quantity of starting material and the minimum working volume of
the TFF system i.e no aggregation was observed under all conditions
examined For both vectors, mono-disperse, non-aggregated vector
was documented immediately following concentration, as well as
after storage at 2-8 °C, and following freeze (-80 °C) / thaw cycling
Together these data support that, while AAV2 vectors are prone to
aggregation at concentrations exceeding the range 1-2 x1013 vg/mL
in parenteral compatible buffers, recombinant AAV8 and 9 -based
vectors can be prepared in neutral physiological buffers at much
higher concentrations, facilitating gene transfer applications requiring
high dose administration in limited volumes
548 Enhanced Fracture Healing Utilizing
Directed Evolution of Adeno-Associated Virus
Seth G Levy,1 Damien Marsic,3 Shawn Gilbert,2 Sergei
Zolotukhin,3 Selvarangan Ponnazhagan.1
1 Pathology, The University of Alabama at Birmingham,
Birmingham, AL; 2 Surgery, The University of Alabama at
Birmingham, Birmingham, AL; 3 Pediatrics, University of Florida,
Gainsville, FL.
Recombinant adeno-associated virus (rAAV) has gained much
attention for its lack of pathogenicity as a gene transfer vector With
up to 10% of the 6 million fractures each year in the US resulting in a
delayed healing or failure to form bony union (non-union), rAAV can
be exploited to potentially deliver therapeutic stimuli to the fracture
site through gene transfer Nonetheless, targeted enrichment of rAAV
at fracture sites in particular of bone progenitor cells has been a
limitation to this concept To overcome this and develop rAAV therapy
for non-union fractures, we have employed an in vitro selection
process for generating bone progenitor cell-specifi c AAV capsids
using a novel capsid library, consisting of random peptide insertion
constructed through isothermal DNA assembly The library containing
heterogenome capsid mutants were selected for in in vitro evolution
in mouse mesenchymal stem cells Capsid mutants recovered from
several rounds of biopanning were analyzed by sequencing The
novel mutants will be further characterized for osteoprogenitor cell
transduction in vitro and for effi cacy of therapeutic gene delivery in
vivo for non-union fractures in an immunocompetent mouse model
549 Targeted Bio-Engineering of Adeno Associated Virus Serotype rh.10 Capsid Improves
Its Gene Delivery In Vitro and In Vivo
Ruchita Selot,1 Nishanth Gabriel,2 Monika Kumari,1 Giridhara R Jayandharan.1,2
1 Center for Stem Cell Research, Christian Medical College, Vellore, India; 2 Hematology, Christian Medical College, Vellore, India.
AAVrh.10, a Clade E primate AAV has favorable biological properties as a vector for neonatal liver-directed gene transfer, including higher levels of transgene expression and vector copy-number persistence during the period of rapid neonatal growth In addition this isolate has the ability to transverse blood-brain-barrier and presumably has the lowest level of circulating neutralizing antibodies against any AAV serotype in humans In the present study,
we investigated if targeted mutagenesis of AAVrh.10 viral capsid residues in a group of lysine (K>R), serine (S>A) and threonine (T>A) residues can improve AAVrh.10 mediated gene delivery This is based on our previous observation in AAV2 vectors, where modifi cations within the phospho S, T residues and ubiquitinable
K residues that reside in close vicinity (9-13 residues in spatial proximity) and form phosphodegron motifs recognized by ubiquitin ligases was benefi cial We fi rst introduced single mutations on the AAVrh.10 capsid at 9 sites (K84R, K137R, K333R, S498A, S501A, S671A, T108A, T252A, T674A) Highly purifi ed stocks of self-complementary wild-type (WT) AAVrh.10 or the mutant AAVrh.10 vectors containing the enhanced fi refl y luciferase gene driven by the chicken -actin promoter were generated by polyethyleneimine based triple transfection of AAV-293 cells The packaging titers of all these vectors were comparable (1x1011vg/ml to 3.2x1012vg/ml) The transduction effi ciency of these mutant AAVrh.10 vectors was
then tested in vitro in human cervical carcinoma cell line (HeLa)
at an MOI of 5000 Forty-eight hours post infection, the transgene expression was determined Amongst all mutant vectors tested, the AAVrh.10- K333R, T674A and S671A vectors demonstrated 4-6 fold higher transduction effi ciency in comparison to AAVrh.10- WT
vector (Fig 1) To further determine their effi ciency in vivo, groups of
C57BL6/J mice were injected with 5x10^10 vgs per animal of either the AAVrh.10- WT or the AAVrh.10S671A vector Our results show that two weeks post gene transfer, the AAVrh.10-S671 vectors exhibit (>2-fold) higher transgene expression in comparison to AAVrh.10-WT vectors Long-term follow up data from these animals will further demonstrate the effi ciency, safety and bio-distribution profi le of these novel AAV rh.10 vectors