CLINICAL GENE THERAPY ORAL ABSTRACT SESSION
359. Gene Therapy for Limb Girdle Muscular
Dystrophy Demonstrates Safety and Improves
Muscle Fiber Size
Jerry R. Mendell,1 Louise R. Rodino-Klapac,1 Xiomara Rosales-
Quintero,1 Janaiah Kota,1 Brian D. Coley,1 Gloria Galloway,1
Josepha M. Craenen,1 Sarah Lewis,1 Vinod Malik,1 Christopher
Shilling,1 Barry J. Byrne,1 Thomas Conlon,1 Katherine J.
Campbell,1 William G. Bremer,1 Laurence Viollet,1 Christopher M.
Walker,1 Zarife Sahenk,1 K. Reed Clark.1
1
The Research Institute at Nationwide Children’s Hospital,
Columbus, OH.
Limb-girdle muscular dystrophy (LGMD) type 2D is defined
as deficiency of a-sarcoglycan (α-SG), a sarcolemmal protein
contributing to membrane stability. Overall, the clinical spectrum
associated with α-SG deficiency overlaps in severity with Duchenne
and Becker muscular dystrophies. Gene replacement strategies have
been challenged by pre-clinical findings related to the cytotoxicity
of α-SG overexpression using adeno-associated virus (AAV) and
cytomegalovirus immediate early promoter/enhancer. In contrast,
prolonged gene expression was seen using a muscle specific promoter.
With this in mind, a double-blind, randomized controlled trial was
initiated in LGMD2D using rAAV1 with full length human α-SG
cDNA (SGCA) under control of a truncated muscle creatine kinase
(tMCK) promoter. The extensor digitorum brevis (EDB) muscle
received 3.25 X 1011 vector genomes on one side, while the control
side received saline. The study was designed for dose escalation but
robust gene expression, without adverse events, negated the need to
enroll a second cohort. All analyses were done prior to breaking the
blind. The number of α -SG positive fibers reached 57% (Subject 1),
69% (Subject 2) and 62% (Subject 3) in the three subjects. Western
blot showed a 4 to 5 fold increase in each subject with a maximum
restoration of α-SG in any block of 76% of normal muscle. Subject
2 demonstrated sustained gene expression for 3 months without
reduction. On the side of increased α-SG expression, Taqman
Quantitative PCR confirmed gene transfer using primers unique to
the α-SG cassette. SGCA transgene copies increased by an average
of 48-fold over baseline (representing 3.6 copies per nucleus). After
persistent gene expression for 3 months, Subject 2 showed an increase
in mean fiber diameter (32.4 ±12.9 mm to 45.4±10.2 mm, p < 0.001).
The total numbers of CD8+ and CD4+ cells per mm2 area was not
different between sides. A few scattered clusters of mononuclear
cells were seen after 3-months that did not affect gene expression.
Serial ELISpot assays specific for IFN-γ secretion beginning pre-gene
transfer showed no response in subjects 2 and 3 to α-SG or AAV1
capsid peptide pools. Subject 1 had a transient minimal response
to Pool 2 of the AAV1 capsid at days 14 and 43. Expansion of
mononuclear cells by capsid peptide pool restimulation with assays
to detect low level anti-AAV capsid responses failed to detect IFN-γ
secretion. At no time was there evidence of a T cell response to the
transgene product, α-SG. No anti-α-SG antibodies were detected for
any subject. This first promising trial of gene therapy for muscular
dystrophy demonstrated robust gene expression persisting for 3
months using a muscle specific promoter. Increased muscle fiber size
favors efficacy of gene transfer. The study lays the foundation for
long-term gene expression studies and vascular delivery trials.
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360. Capsid Antigen Presentation Flags
Hepatocytes for Destruction after Transduction by
Adeno-Associated Virus
Gary C. Pien,1 Etiena Basner-Tschakarjan,2 Daniel J. Hui,2 Ashley
Mentlik,1 Jonathan D. Finn,2 Nicole C. Hasbrouck,2 Shangzhen
Zhou,2 Samuel L. Murphy,2 Marcela V. Maus,2 Federico Mingozzi,2
Jordan S. Orange,1 Katherine A. High.2,3
1
Division of Allergy and Immunology, Children’s Hospital of
Philadelphia, Philadelphia, PA; 2Department of Hematology,
Children’s Hospital of Philadelphia, Philadelphia, PA;
3
Howard Hughes Medical Institute, University of Pennsylvania,
Philadelphia, PA.
Adeno-associated virus (AAV) vectors are effective gene delivery
vehicles mediating long-lasting transgene expression. Data from a
clinical trial of AAV2-mediated hepatic gene transfer of factor IX
(F9) into hemophilia B subjects suggested that CTL responses against
AAV capsid eliminated transduced hepatocytes and prevented long-
term F9 expression. However, the capacity of hepatocytes to present
AAV capsid-derived antigens has not been formally demonstrated,
nor whether transduction by AAV sensitizes hepatocytes for CTL-
mediated destruction. We therefore engineered a soluble TCR for
the specific detection of capsid-derived peptide:MHC I (pMHC)
complexes. TCR-multimers exhibited preserved antigen- and HLA-
specificity, and possessed high binding affinity for cognate pMHC
complexes (KD ∼0.26 nM). By using this reagent, capsid antigen
presentation was detectable by confocal microscopy following
AAV-mediated transduction of hepatocytes. Although the number
of pMHC complexes appeared relatively low, it was nevertheless
sufficient to trigger reporter gene expression in a transgenic T cell line
expressing our TCR antigen-binding domains. Moreover, transduced
cells were flagged for destruction by capsid-specific CTLs expanded
from normal human donor PBMCs. Cytolysis of hepatocytes was
inhibited in the presence of our soluble TCR, demonstrating a possible
application for this reagent in blocking undesirable CTL responses.
Together, these studies provide a mechanism for the loss of transgene
expression and transient elevations in aminotransferases following
AAV-mediated hepatic gene transfer in humans, and a potential
therapeutic intervention to abrogate these limitations imposed by
the host T cell response.
361. The Role of the Complement System in
Host Responses to AAV Vector Administration
Gregory S. Arnold,1 Linda L. Burianek,1 Anne K. Zaiss,2 Daniel A.
Muruve,2 Jeffrey S. Bartlett.1,3
1
Gene Therapy Center, The Research Institute at Nationwide
Children’s Hospital, Columbus, OH; 2Department of Medicine,
University of Calgary, Calgary, AB, Canada; 3Department of
Pediatrics, The Ohio State University, Columbus, OH.
The complement system, by virtue of its dual effector and priming
functions, is a major host defense against pathogens. While AAV
vectors are associated with relatively mild host innate responses,
neutralizing antibodies against the vector capsid and transgene still
occur. Here we show that the complement system is integral to
both of these observations. To understand further the basis of the
antiviral immune response to AAV vectors, studies were performed
to characterize AAV interactions with macrophages. While primary
macrophages and macrophage cell lines are very poorly transduced
by AAV, AAV vectors readily bind to and are taken up by these cells
in a receptor-dependent manner. An assessment of internalized vector
genomes showed that vector uptake was enhanced in the presence of
complement components and that enhanced uptake in the presence
of complement coincided with increased macrophage activation as
determined by the expression of NFκB-dependent inflammatory
genes. Further studies demonstrated the binding of human C3
Molecular Therapy Volume 17, Supplement 1, May 2009
Copyright © The American Society of Gene Therapy
 RNA VIRUS VECTORS II: VECTOR DESIGN AND INTEGRATION PROPERTIES
complement fragments to AAV vector particles. Nevertheless, AAV
particles did not activate the alternative pathway of the complement
cascade as suggested by this interaction. Furthermore, AAV lacked
cofactor activity for factor I-mediated degradation of C3b to iC3b.
Instead, our results suggest that the AAV capsid binds the complement
regulatory protein factor H (fH) and that AAV-bound fH mediates
cleavage and inactivation of C3b to iC3b. Thus, AAV capsids bind and
recruit the complement regulatory protein fH, resulting in decreased
complement activation in solution and inactivation of particle bound
C3 fragments. Furthermore, generation of iC3b-modified AAV
vector particles define host interactions based on the distribution of
cellular iC3b receptors. Interestingly, these receptors mediate the
phagocytic clearance of iC3b opsinized AAV vector particles in the
absence of inflammatory signaling and together with inhibition of
complement activation by surface-bound fH account for the mild
inflammatory responses observed in vivo with AAV vectors. While
we have previously shown that the complement system is an essential
component of the host adaptive immune response to AAV. Here we
define an interaction between AAV and the complement system that
attenuates host innate responses. We show that AAV binds to serum
fH and promotes the fH-mediated cleavage of C3b. Because the
AAV capsid lacks any sequence homology to known complement
regulatory proteins, AAV appears to have evolved a novel viral
immune evasion activity akin to that utilized by West Nile virus NS1,
whereby the inflammatory and effector functions of complement are
inhibited by recruiting and binding serum fH.
RNA Virus Vectors II: Vector Design and
Integration Properties
362. Cell-Specific Transcriptional Regulatory
Domains Attract γ-Retroviral Integration in the
Human Genome
Claudia Cattoglio,1 Barbara Felice,2 Davide Cittaro,2 Danilo
Pellin,3 Alessandro Ambrosi,3 Ermanno Rizzi,4 Gianluca De
Bellis,4 Chiara Bonini,1 Giulietta Maruggi,5 Francesca Miselli,5
Manfred Schmidt,6 Lucilla Luzi,2 Alessandra Recchia,5 Fulvio
Mavilio.1,5
1
H. San Raffaele Scientific Institute, Milan, Italy; 2FIRC Institute
of Molecular Oncology, Milan, Italy; 3CUSSB, Vita-Salute San
Raffaele University, Milan, Italy; 4CNR, Milan, Italy; 5University
of Modena and Reggio Emilia, Modena, Italy; 6National Center for
Tumor Diseases, Heidelberg, Germany.
Gene transfer vectors derived from γ-retroviruses target at high
frequency genes involved in the control of growth and differentiation
of the target cell, and may induce insertional tumors or pre-neoplastic
clonal expansions in patients treated by gene therapy. The gene
expression program of the host cell appears instrumental in directing
γ-retroviral integration, but the molecular basis of this phenomenon is
poorly understood. We recently reported a bioinformatics analysis of
the distribution of transcription factor binding sites (TFBSs) flanking
∼4,000 proviruses in human hematopoietic and non-hematopoietic
cells. We showed that γ-retroviral, but not lentiviral, vectors integrate
in genomic regions enriched in cell-type specific subsets of TFBSs.
Analysis of sequences flanking the integration sites of Moloney
leukemia virus (MLV)-derived vectors with modified long terminal
repeats (LTRs), and of a human immunodeficiency virus (HIV)-based
vector packaged with the MLV integrase, showed that the MLV
integrase and LTR enhancer are the viral determinants of the selection
of TFBS-rich regions. The study indicated that γ-retroviruses have
evolved a peculiar strategy to interact with the host cell chromatin,
based on a cooperation between the integrase and TFs bound to
the LTR enhancers before integration to tether pre-integration
complexes (PICs) to transcriptionally active regulatory regions.
To further explore the link between cell-specific transcription and
Molecular Therapy Volume 17, Supplement 1, May 2009
Copyright © The American Society of Gene Therapy
MLV integration, we performed deep sequencing of MLV insertions
in human CD34+ hematopoietic cells (n=33,162), T lymphocytes
(n=7,996), and skin keratinocytes (n=3,020). Compared to matched
random distributions, MLV integrations are highly clustered along
the chromosomes, with peaks of frequency around transcriptionally
active loci. Genes within or near the insertion clusters are shared in
part among the three cell types, but are mostly involved in tissue-
specific development, differentiation and functions, and differentially
expressed, as evaluated by gene expression arrays. Integration
peaks overlap with non-coding elements highly conserved among
mammals, again meaning that genomic regions presumably involved
in transcriptional regulation are attractive for MLV PICs. The status
of chromatin around retroviral and random sites was checked by
ChIP-on-chip technology, revealing marks of active transcription
(H3K9ac, H3K4me2, H3K4me3) on regions flanking the sole MLV
insertions. Taken together, our observations state an overt preference
of γ-retroviral vectors for genomic regions actively engaged in
transcriptional regulation. We speculate that MLV PICs are directed to
these regions by interaction with general components of the enhancer-
binding complexes, rather than with specific TFs or TF families.
363. Mouse Models To Assess the Risk of
Vector Insertional Mutagenesis upon Systemic
Delivery
Marco Ranzani,1,2 Daniela Cesana,1,2 Manfred Schmidt,3 Francesca
Sanvito,4 Fabrizio Benedicenti,1 Cynthia Bartholomä,3 Maurilio
Ponzoni,4 Alessio Cantore,1,2 Lucia Sergi Sergi,1 Claudio Doglioni,4
Christof VonKalle,3 Luigi Naldini,1,2 Eugenio Montini.1
1
HSR-TIGET, Milan, Italy; 2San Raffaele University, Milan, Italy;
3
NCT, Heidelberg, Germany; 4HSR, Milan, Italy.
Lentiviral vectors (LV) can transduce a broad spectrum of non-
dividing cells in vivo including hepatocytes. Efficient liver gene
transfer and long term transgene expression may allow the treatment
of several hepatic and systemic diseases. However, vector integration
may occasionally lead to transformation of hepatocytes, as it has been
reported for AAV and non-primate LV. Therefore, sensitive preclinical
models to assess the genotoxic potential of vector integration in liver
tissue are necessary to validate the safety of liver gene transfer. We
previously developed a sensitive in vivo genotoxicity assay based
on transduction/transplantation of hematopoietic stem/progenitor
cells (HSPC) from tumor prone Cdkn2a-/- mice. By this approach
we demonstrated that LV with self-inactivating (SIN) long terminal
repeats (LTR) are less genotoxic than γ-retroviral vectors and that
genotoxicity is strongly modulated by the vector design and the
integration site selection of each vector type. However, the predicted
safety of an LV design validated in HSPC may not necessarily apply
to the liver tissue. To setup sensitive assays for liver cell genotoxicity
in vivo, we exploited two tumor-prone mouse models and a tumor-
promoting regimen applied to wild type mice after vector injection.
We validated these models by challenge with LV engineered with
powerful enhancers/promoters in the LTR previously shown to
be genotoxic in HSPC. Temporal vein injection of this vector in
newborn Cdkn2a-/- mice induced early onset of histiocytic lymphomas
infiltrating the liver and spleen with respect to uninjected mice
(p<0.0001). These data show that in this tumor prone model, non-
hepatocyte cells are the most susceptible target for cell transformation
upon systemic injection of a genotoxic LV. On the other hand,
injection of an LV with powerful hepatospecific enhancers in the LTR
(LV.ET.LTR), induced high grade hepatocellular carcinomas (HCC) in
30% of mice, whereas none was found in untreated controls. In 50%
of the HCCs (4/8) LV integrations clustered in a 2.5kb region within
the Braf oncogene. In these HCCs we detected chimeric LV-Braf
fusion transcripts encoding for a putative truncated Braf protein with
constitutive kinase activity that has been directly implicated in cell
transformation. Moreover, LV.ET.LTR treatment induced liver tumors
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