374 optimization of AAV vectors for improved therapeutic protein expression in the canine models for duchenne muscular dystrophy

374 Optimization of AAV Vectors for Improved Therapeutic Protein Expression in the Canine Models for Duchenne Muscular Dystrophy Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Ame[.]

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S144

Similar results were obtained when a tyrosine triple-mutant AAV2

vector was used Western blot analyses with cytosolic and nuclear

fractions prepared from transduced primary human DCs further

corroborated that the alternative pathway of NF-kB activation

(accumulation of p100 and p52 proteins) was operational (Fig

1B) Additional studies revealed high levels of expression of DC

maturation markers, CD83 and CD86, which were inhibited by an

NF-kB inhibitor, Bay11 In preliminary experiments, shown in Fig

2, the levels of interferon-gamma (IFNγ) produced in response to

transduction by the tyrosine triple-mutant vectors (green arrowheads)

were lower than those by the wild-type AAV vectors (red arrowheads),

although the data did not reach signi cance (Fig 2, Panel A)

Differences in the CTL response against EGFP with the

triple-mutant vector, on the other hand, were signi cant (Fig 2, Panel B)

We speculate that the use of NF-kB inhibitors and/or transduction with

the triple-mutant vectors would lead to dampened host cell immune

response in general, and cytotoxic T cell response in particular, against

AAV vectors, which has signi cant implications in the optimal use

of these vectors in human gene therapy

371 Optimizing Capsid Composition for the

Production of Recombinant Adeno-Associated

Virus Using the Baculovirus Expression System

Kirsten Erger,1 David Markusic,1 Ramaz Geguchadze,1 Barry J

Byrne,1 Sergei Zolotukhin.1

1 Pediatrics, University of Florida, Gainesville, Fl.

The scalability and  exibility of recombinant AAV vectors has

been expanded by the production of rAAV in insect cells using the

recombinant baculovirus system In this system, Sf9 insect cells

grown in suspension are co-infected with three baculovirus expression

vectors (BEVs): BacRep, BacVP, and Bac-GOI (gene of interest

 anked by AAV inverted terminal repeats) encoding the respective

components of the rAAV production machinery Recently, we have

described a simpli ed version of the baculovirus system where

multiple copies of rep and cap helper genes were stably integrated

into the Sf9 cell chromosome and the production cycle was initiated

by an infection with a single BEV encoding rAAV cassette Here, we

extend the utility of the described stable cell lines to produce rAAV

of alternative serotypes (such as AAV8) characterized by higher

transduction properties

One of the shortcomings of the Baculovirus system is the low

infectivity of some AAV serotypes (e.g AAV8 and AAV5) due to

the low content of VP1 capsid protein containing phospholipase

A2 activity Presumably, a non-canonical ACG start codon in some

sequence context is not as ef cient as within AAV2 sequence

Therefore, the canonical ATG start codons for the VP1 capsid genes

of AAV2 and AAV8 were re-introduced These plasmids were mixed

with the respective ACG-containing vector genomes at the different

stoichiometric ratios using transient transfection, the resultant

virions contained higher ratios of VP1 and reduced amounts of VP2

Surprisingly, a doublet band at the position of what was supposed to

be VP3 was also seen An increase in the higher molecular weight

band with a concurrent decrease in the lower band was seen as the

ratios for the ATG VP1 increased The proteomics analysis had shown that the higher band was a cleaved version of VP1 and/or VP2, while

the lower band was in fact VP3

We further posited that an increase in VP1 content incorporating PLA2 domain will result in more infectious vector To prove this hypothesis, we derived Sf9 stable cell lines expressing AAV2 and AAV8 capsid proteins Based on transient expression pilot experiment,

we selected the input helper plasmids ratios either ACG:ATG=2:1 for AAV2 or 1:1 for AAV8 Stable cell lines containing rep and cap were developed and the capsid ratios were con rmed by inducing their expression with Bac-rAAV-GFP The Western blotting analysis

of the induced integrated cap genes con rmed the selected ratios

Their compositions were similar to those produced in HEK 293 cells while very distinct from ACG only-derived vectors

The AAV8 stable cell lines were used to produce rAAV8-GFP vector by infection with the Bac-rAAV-rAAV8-GFP helper In vivo experiments with these vectors showed an increase in transduction ef ciency as compared to rAAV8 vectors produced without mosaic VP1 capsid, which were essentially noninfectious Additionally, the transduction ef ciency of baculovirus produced rAAV8-GFP seemed

to be as good or greater compared to rAAV produced in HEK 293 cells

This modi ed system extends the usability of AAV vector production using the baculovirus expression system

372 Genetically Re-Engineered AAV Rep78 Identi es Sequence-Restricted Inhibitory Regions

Affecting Adenoviral Replication

Varsha Sitaraman,1,2 Patrick Hearing,2 Steffen Mueller,2 Charles Ward,3 Dmitri Gnatenko,1 Steven Skiena,3 Eckard Wimmer,2 Wadie

F Bahou.1

1 Medicine, SUNY Stonybrook, Stony Brook; 2 Molecular Genetics &

Microbiology, SUNY Stonybrook, Stony Brook; 3 Computer Science, SUNY Stonybrook, Stony Brook.

Hybrid Adenovirus/Adeno-associated viruses (Ad/AAV) combine Ad’s capacity, tropism and ease of production with AAV’s ability for site-speci c integration (SSI) into chromosome 19 AAVS1 The AAV Rep78 protein required for SSI, displays an inhibitory effect on Ad replication, particularly when co-expressed within the Ad backbone

We applied a computational algorithm to alter the 1.8Kb Rep78 gene, hypothesizing that its apparent cis-acting inhibitory effect could be attenuated by modifying its DNA sequence Two distinct

genes were synthesized de novo:(1)scrambled Rep78 (sRep78)

which incorporated changes by randomly mixing synonymous codons and (2)codon-deoptimized Rep78 (dRep78) in which, based

on codon pair bias, synonymous codon exchanges were restricted to underrepresented codon pairs (codon pair bias refers to the preference for some codon pairs over other synonymous codons to encode the same pair of adjacent amino acids Utilization of underrepresented codon pairs results in an ORF that is expressed at lower levels)

sRep78 and dRep78 were 70% and 80% similar to the wild-type (wtRep78) nucleotide sequence, but encoded exactly the same amino acid sequence as wtRep78 As con rmed by immunoblot analysis

in transfected HeLa cells, sRep78 was identical in expression level

to wtRep78 and dRep78 was expressed at only ∼50% In contrast to the similar amounts of Rep78 protein between wtRep78 and sRep78, there was a dramatic difference in Ad replication capacity when the constructs were expressed under a tetracycline-inducible promoter within E1-deleted recombinant Ads Also, in spite of the difference in protein expression, at all passages studied both Ad/dRep78 and Ad/

sRep78 demonstrated essentially equivalent and normal replication rates and stability when propagated in 293 cells, with viral particle(p) titers comparable to other E1-deleted Ads lacking Rep78 (Ad/

dRep78– 2.8x1012p/mL; Ad/sRep78– 1.32x1012p/mL; Ad/FVIII–

1.88x1012p/mL); these results sharply contrasted with those using Ad/

wtRep78 where viral replication remained inhibited at all passages To

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S145

con rm the integrity of Rep activity, excision assays were completed

in 293 cells coinfected with either Ad/dRep78 or Ad/sRep78, and an Ad/AAV virus expressing the human Factor VIII gene  anked by the AAV terminal repeats As expected, a single ∼8Kb excision product was clearly evident using either virus, con rming the functionality

of the dRep78 and sRep78 proteins; further, nearly 3-fold greater excision was seen in the presence of inducer (1µg/ml doxycycline)

In summary, these data strongly suggest that Rep78 sequences inhibit

Ad replication unrelated to Rep78 protein function, and identify novel strategies for maintaining and propagating Rep78 within single-backbone Ad/AAV viruses for potential site-speci c transgene delivery Ongoing structure/function studies using computational methods that build on these initial observations will likely re ne the speci c sequences and molecular mechanism(s) of this effect

373 Improved and Persistent Expression with Novel AAV Serotypes for Neonatal Gene Therapy

Chuhong Hu,1 Fides D Lay,1 Joseph Springer,1 Ronald W

Busuttil,1 Gerald S Lipshutz.1

1 Surgery, UCLA School of Medicine, Los Angeles, CA.

Neonatal gene therapy is a promising strategy for treating a number

of congenital diseases that can be diagnosed either prenatally or shortly after birth Correction of genetic diseases during the neonatal period may be achieved by targeting gene replacement therapies to expanding stem cell populations and to developing organ systems and

if succsessful, could limit or abrogate the pathologic consequences

of genetic mutations However, stable expression in the face of rapid cell division and growth may lead to failure of long-term therapy The goal of these studies was to examine the distribution of and level of expression into early adulthood of 10 natural serotypes of AAV when

administered to the rapidly growing neonate Methods: We developed

10 rAAV serotypes (1-rh10) with the chicken β-actin promoter/CMV enhancer and  re y luciferase cDNA 2x10e10 genome copies of rAAV was administered intravenously to 2-day-old mice Expression was followed for the  rst 100 days of life both by whole animal bioluminescent imaging (BLI) and by tissue luminometry at selected

time points Vector copy number was determined by qPCR Results:

Mouse growth was particularly rapid during the  rst 5 weeks of life with a 3.5-fold increase in weight from 1.7±0.2 grams at birth to 17.6±2.9 grams at 5 weeks Subsequently, mice grow much more slowly weighing 21.3±2.3 grams at day 50 and 29.0±1.2 at day 100 (males) All ten serotypes demonstrate expression within 72 hours

of intravenous administration By whole animal BLI, AAV can be subdivided into high- (8,9 rh10), medium- (1, 5, 6, 7), and low-expressing (2, 3, 4) serotypes in the neonate The lowest low-expressing serotype at all time points was AAV3 and the highest was AAVrh10

Liver expression was highest with rh10>>8>9>7>6>1 Cardiac expression was highest with rh10>9>8>7=6=1 Skeletal muscle expression was highest with rh10>9>8>7>6=1 In general, the rh10 serotype provided the highest expression 3 days after injection and remained highest at 100 days of life In most organs, including the liver, there is a rapid decline in level of expression over time, likely

in part related to cellular division However in skeletal muscle near terminal differentiation, expression is stable or increases with animal growth

Conclusions: Neonatal gene therapy is challenged by the need

to transduce rapidly growing and dividing cells as the organism develops Long-term expression in rapidly dividing tissues can be obtained with AAV as stable expression occurs after the rapid period

of growth is completed In general this was best obtained in tissues with serotype rh10 However, in tissues where growth is a greater feature than cellular division (i.e skeletal muscle), stable or increasing

expression is obtained early after AAV administration.

374 Optimization of AAV Vectors for Improved Therapeutic Protein Expression in the Canine Models for Duchenne Muscular Dystrophy

Alock Malik,1 Marilyn Mitchell,1 Andrew Mead,1 Mike Petrov,1

Hansell Stedman.1

1 University of Pennsylvania, Philadelphia, PA.

A major bottleneck in the development of effective gene therapy for Duchenne Muscular Dystrophy is the design of vectors that yield the highest possible expression of dystrophin or utrophin in the target tissues The magnitude of this challenge has come into clearest focus during the transition from small to large animal models in the preclinical setting We recently initiated studies of limb-wide, vascular gene transfer in the dystrophic dog using AAV vectors based on internally deleted versions of the wild type canine dystrophin and utrophin cDNA sequences Early in the course of these studies, the need for higher levels of protein expression normalized

to the administered vector dose prompted a parallel effort to further optimize the entire vector expression cassette Iterative improvements were made through the analysis and serial modi cation of plasmids encoding identical proteins using distinct coding sequence and transcriptional cassettes Systematic screening,  rst in HEK 293 cells and then in mdx mouse muscle allowed us to determine the single best expression cassette from a pool of plasmids with different promoters (ubiquitous, muscle specific and hybrid-synthetic promoters),

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S146

introns, poladenylational signals, transcriptional enhancer elements,

and peptide coding sequences The best combination of these

improvements thus far has provided at least a 10-fold higher level of

recombinant protein expression than our  rst generation cassette, with

further improvements under additional analysis Selected versions of

these transcriptional cassettes are being further characterized in the

context of myotrophic AAV vectors following administration to mdx

mice and GRMD dogs 10-fold or greater improvements due to the

optimization process should greatly accelerate the pace of therapeutic

trials in the dystrophic dogs

375 rAAV1 Infect Mouse Bone Marrow-Derived

Dendritic Cells through Receptors with Į2,3 and

Į2,6 Sialic Acids

Yuanqing Lu, Sihong Song

Pharmaceutics, University of Florida, Gainesville, FL.

Host immune response to recombinant adeno-associated virus

(rAAV) is still a hurdle on the road of gene therapy, although rAAVs

may have low immunogenicity compare with other viral vectors

Better understanding of the mechanism of immune response against

rAAVs is important for the safety and optimal effect of gene therapy

We have recently shown transduction of dendritic cells (DC) plays a

critical role in host immune response to the transgene product of rAAV

vectors Different rAAV serotypes display difference immunogenicity

depending on their ability of DC infection rAAV1 ef ciently infected

DC and induced strong immune responses in several stains of mouse

However, the receptors on DC that interact with rAAV1 vector remain

unknown In this study we investigated the rAAV1 receptor on DC

To obtain mouse DCs, mouse bone marrow (BM) was isolated

from none-obese diabetes (NOD) mice BM cells were cultured in

complete RPMI-1640 medium with 2ng/ml of recombinant murine

granulocyte-macrophage colony stimulating factor and 5ng/ml of

IL-4 for 3days On day4, cells were infected with rAAV1 with 1x104

of MOI Lectin competition assay showed that α2,3 or α2,6 sialic

acids were important for rAAV1 infection of DCs This infection

can be inhibited by treatment of DCs with proteinase K indicating

glycoprotein may be involved In addition, we showed that LPS

pre-treated DCs can not be infected by rAAV1 indicating that rAAV1

unable to infect mature DCs Taken together, these results suggest

that rAAV1 infect mouse bone marrow derived immature dendritc

cells through receptors with α2,3and α2,6 sialic acids

376 Using AAV Vector as Carrier for Biosensor:

Detecting microRNA’s Activities by Reverse

Infection of AAV Vectors

Wenhong Tian,1 Xiaoyan Dong,2 Gang Wang,1 Shuping Tan,1

Xiaobing Wu.1

1 National Laboratory of Molecular Virology and Genetic

Engineering, Institute for Viral Disease Control and Prevention,

Chinese Center for Disease Control and Prevention, Beijing,

China; 2 Beijing Fiveplus Molecular Medicine Institute Ltd.,

Beijing, China.

Objective Recombinant adeno-associated virus (rAAV) vectors

have been used increasingly for gene therapy However, using rAAV

as carrier for a detecting sensor has never been reported Here we

describe a novel strategy of using rAAV carried biosensors to detect

microRNAs activities in life cells named as ‘A-sensors’ A-sensor

is a rAAV containing Gaussia luciferase (Gluc) gene expression

cassette with one copy of miRNA-complementary sequence in its

3’UTR Also a  re y luciferase expression cassette is included as

an inner control for calibrating transduction titers between different

A-sensors Methods To test if cells could be transduced by rAAV in

‘reverse infection’ way, rAAV2-EGFP in serial dilutions were coated

on a cell culture plate and air-dried Different cells were added to the

plate and cultured in CO2 incubator at 37°C and investigated EGFP expression in 24 to 48h A-sensor vector was designed as shown in

Figure1, with BglII and EcoRI site (downstream of Gluc gene) for

inserting miRNA-complementary sequence rAAV was produced and puri ed by our previous methods with rHSV1-repcap as helper virus

A-sensors carrying various miRNA-complementary sequences of equal transduction titer were separately coated on 96-well cell culture plate, and dried A-sensor without any miRNA-complementary sequence was coated as control Different A-sensors were arranged orderly in the culture plate resulting in ‘A-sensors array’ Cells to be detected were added with equal number to wells of the A-sensor plate

The Gluc activities in the supernatant were detected after 24∼48h

The relative microRNA activities could be presented by comparing Gluc activity of the control A-sensor to that of the speci c miRNA A-sensor To verify the method, miR-142 3p was chosen as the  rst

detecting subject Results Results showed rAAV2-EGFP coated on

culture plate in air dried condition transduced various cells effectively and dose dependently Using the A-sensor method, miR-142 3p activity was found high in U937, K562, Sp2/0, and P815 cells, and negative in HEK 293

Conclusion rAAV could transduce cells effectively in a reverse

infection way, showing a great advantage for using AAV vector as carrier for biosensor This study provides a novel strategy for detecting miRNA activity pro les

377 Clearance of Herpes Simplex Virus

by Orthogonal Manufacturing Process of Recombinant Adeno-Associated Virus (rAAV) Vectors

Guo-jie Ye,1 David R Knop,1 Darby L Thomas,1 Lijun Wang,2

Jeffrey D Chulay.1

1 Research & Development, Applied Genetic Technologies Corporation, Alachua, FL; 2 Process Development, Florida Biologix, Alachua, FL.

Purpose: rAAV manufacturing using a recombinant herpes simplex

virus (rHSV) complementation system employs two ICP27 mutant rHSV helper viruses that are replication-incompetent in normal cells but can be propagated in complementing V27 cells (Vero cells stably transformed with the HSV-1 UL54 gene encoding ICP27)

During rHSV production, a replication-defective ICP27 mutant rHSV can recombine with the UL54 gene in the V27 cell resulting in low levels of replication-competent HSV (rcHSV) revertants which may further propagate during rAAV production and carry through into the unprocessed pre-lysis bulk product We evaluated the ability of the current puri cation process used for GMP manufacture of a rAAV vector expressing human alpha-1 antitrypsin (rAAV1-CB-hAAT)

to effectively inactivate and remove all detectable infectious HSV

Methods: A 10 L scale batch of rAAV1-CB-hAAT was initiated for

generation of matrix samples to support viral clearance studies, using the same process used for manufacturing clinical grade rAAV1-CB-hAAT drug substance Three manufacturing unit operations were evaluated for HSV clearance: detergent lysis, CIM-Q anion exchange chromatography, and AVB affinity chromatography Each unit operation was scaled appropriately to mimic “at scale” production

Matrix materials spiked (1/10, v/v) with high-titer wild-type HSV (wtHSV) were subjected to the process unit operations under study and HSV clearance measured using a quali ed HSV infectious titer assay Clearance of HSV was assessed at speci c steps and

cumulatively throughout the manufacturing process Results: The

three unit operations (detergent lysis, CIM-Q and AVB column

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S147

ADENOVIRUS AND OTHER DNA VIRUS VECTORS II

chromatography) achieved HSV clearance of 6.84, 4.27, and 2.73 log10 individually, with a cumulative 13.84 log10 of infectious HSV In a 25

L production batch, the total input quantity of replication-incompetent rHSV is 5 x1011 pfu and the total quantity of residual infectious HSV (including both rHSV and rcHSV) in unprocessed bulk product before detergent lysis is < 2.5 x109 pfu A further 13.84 log10 reduction of this value from the subsequent detergent lysis and chromatography steps results in a theoretical <3.6 x10-5 pfu of infectious HSV remaining

in the  nal puri ed bulk product This would imply that the highest planned single dose administered to a patient would contain <6.5 x10-5

pfu of infectious HSV, a safety margin of >4 log10 Conclusion: The

cumulative clearance of infectious HSV by the three process steps in the AAV1-CB-hAAT manufacturing process is estimated to be ≥13.8 log10, indicating there is a > 4 log10 safety margin that no HSV will

be present in the highest planned patient dose

Adenovirus and Other DNA Virus Vectors II

378 Production of a Novel Class of Human Polyomavirus JC Vectors Using Baculovirus Expression System

Kirsten E Erger,1 Oleg Gorbatyuk,2 George Aslanidi,1 Ramaz Geguchadze,1 Barry J Byrne,1 Sergei Zolotukhin.1

1 Pediatrics, University of Florida, Gainesville, FL; 2 Molecular Genetics and Microbiology, University of Florida, Gainesville, FL.

Myelinating cells, oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS) are cell populations responsible for demyelination disorders such as Canavan disease and multiple sclerosis, an autoimmune disorder of the CNS Currently, there is no effective therapy for patients with disorders of these cell populations Treatment of such neurodegenerative diseases by gene therapy may require vectors characterized by enhanced tropism towards myelinating cells So far, attempts to derive such vectors have been met with limited success

JCV, also known as polyomavirus JC, is a member of a family

of small nonenveloped DNA tumor viruses that incorporate small, circular, double-stranded DNA genomes of about 5 Kbp In humans, JCV infects oligodendrocytes and astrocytes and approximately 70-90% of the adult population is seropositive In patients with suppressed immune system, JCV induces a demyelinating disease – progressive multifocal leukoencephalopathy (PML) In this report,

we describe a new system for a scale-up production of recombinant JCV vector The system includes genetic components of three un-related viruses: Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), Adeno-Associated Virus (AAV), and JCV Stable insect cell lines incorporate helper genes coding for JCV capsid proteins VP1/2/3, and AAV Rep In addition, cell lines incorporate transgene cassettes devoid of prokaryotic replication origin and antibiotic resistance genes Instead, the cassettes include baculovirus and AAV replication origins, a baculovirus enhancer homologous region 2 (hr2) and an AAV inverted terminal repeat (ITR) sequence

Integrated helper genes are designed to remain silent until the cell is infected with baculovirus expression vector (BEV) which provides the immediate-early (IE-1) transcriptional transregulator Infection with BEV initiates the rescue/ampli cation of the integrated helper genes and the transgene cassette A circular, double stranded replicating transgene cassette emulates the JCV genome, allowing its ef cient packaging into JCV capsids

The initial characterization of the system including recombinant JCV (rJCV) puri cation, yield, capsid protein stoichiometry, and particle-to-infectious particle ratios will be discussed In vivo data obtained in rat’s brain will be provided

The described novel JCV vector  lls the void among existing vectors by its natural tropism towards previously inaccessible cell populations – oligodendrocytes and astrocytes in the human brain This

report validates the notion that insect stable cell lines incorporating highly inducible helper genes can be utilized as a universal platform for the assembly of variety of mammalian viral vectors

379 Induction of Cell Surface Integrins in Lymphomas and Potential Applications to Gene Therapy

Ann Marie O’Neill,1 Farruk M L Kabir,1 David T Curiel,3 Laura Timares,4 Bruce F Smith.1,2

1 Scott Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL; 2 Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL; 3 Departments of Medicine, Obstetrics and Gynecology, Pathology, Division of Human Gene Therapy, University of Alabama, Birmingham, AL; 4 Departments of Dermatology, Cell Biology, Division of Human Gene Therapy, University of Alabama, Birmingham, AL.

The successful development of a gene therapy approach to B cell lymphoma requires the construction of vectors that speci cally target cancer cells and the use of an appropriate model for evaluation of these vectors In this regard, canine lymphoma is an excellent model

of human non-Hodgkins Lymphoma The canine disease has a similar etiology and presentation to the human disease, the model has an intact immune system and the size of the animal model allows for extrapolation to humans Recombinant adenovirus vectors (Ad) have

been recognized as an effective in vivo gene delivery system and

have been utilized as gene therapy agents for a number of cancers The elucidation of the mechanics of viral entry has allowed the development of recombinant vectors that exploit existing cell surface receptors to achieve viral entry into the cell B cells are normally resistant to infection by Ad5, likely due to the lack of the Coxsackie and Adenovirus receptor (CAR) on the cell surface and low level expression of the αvβ3/5 integrins necessary for viral internalization Our analysis of primary canine lymphoma cells has indicated that these cells resemble lymphocytes with respect to a paucity of CAR and integrin expression Ad5 vectors carrying the luciferase gene with

 ber modi ed to incorporate an RGD motif, which binds integrins,

a pk7 motif, which binds heparan sulfate containing receptors, or doubly modi ed to include both were used to infect a control cell line,

a canine lymphoma cell line (OSW) and primary canine lymphoma cells Both the Ad5 pk7 and Ad5 RGD vectors were able to ef ciently transduce control cells In contrast, none of the Ad5 vectors was able to transduce unstimulated lymphoma cells The level of cell surface expression of αvβ3 integrin was measured on resting OSW cells and primary lymphoma cells OSW cells were then treated with LPS, ConA or PMA plus ionomycin and then re-examined and integrin expression was shown to increase in response to stimulation leading to the potential of enhanced Ad infection rates Blocking of integrin reduced transduction Previous studies have indicated that

Ad can be successfully retargeted to cell surface receptors other than CAR, however low level expression of integrins on lymphoma cells appears to limit the ef ciency of transduction by retargeted Ad in these cells Our experiments to date suggest that native viral tropism can be modi ed but canine lymphoma cells express low levels of

an integrin necessary for virus internalization This has important implications with regard to targeting strategies as a paucity of integrin internalization receptors remains a limiting factor