107 isolation and characterization of the heparin binding domain of AAV

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Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

S42

BIOLOGY OF TRANSDUCTION

the binding of cellular proteins to the double stranded D (dsD)

sequence of the adeno-associated virus (AAV) inverted terminal

repeat (ITRs) by electromobility shift assay We show here that

transcription factors belonging to the RFX family, bind specifically

and selectively to AAV2 and AAV1 dsD sequences By supershift

experiments with antisera against RFX family members and using in

vitro translated RFX proteins, we further show that the observed

complexes, contain RFX1 homodimer and RFX1/RFX3

heterodimers RFX proteins, which interact with transcriptional

enhancers and origins of replication of several mammalian DNA

viruses including Hepatitis B, Polyoma and Cytomegalovirus, may

therefore be involved in the replication cycle of AAV

105 Use of a Recombinant Adenovirus

Expressing the AAV-2 Rep52 Helicase To Enhance

Vector Potency from Stable rAAV Producer Cell

Lines

Chun-Liang Chen, Ruju Chen, Philip R Johnson, K Reed Clark

1 Center for Gene Therapy, Columbus Children’s Research

Institute, Columbus Children’s Hospital, Columbus, OH.

Background To augment yields from stable rAAV producer cell

lines, we previously down-modulated Rep78/68 protein levels by

replacement of the native p5 rep promoter with the tetracycline

responsive element (TRE) Following wild-type Ad5 infection,

rAAV/Β-gal producer cells containing this modification yielded

6-10 times more infectious rAAV than p5 containing lines The

modified cell lines also demonstrated an increase in Rep52/40

expression levels Southern blot analysis revealed that purified

vector particles derived from these Rep modified cell lines possessed

a greater proportion of full-length vector genomes (4 to 8-fold)

Moreover, a direct role for Rep52/40 function in this process was

suggested by transient transfection experiments with a Rep52/40

expression plasmid, whereby enhanced vector potency was observed

in the transfected cells To apply these observations towards a

scalable production system, we report the isolation of a replication

competent, adenovirus type 5 vector that expresses the Rep52

protein The use of this virus in pilot production experiments is

described below Methods The recombinant Ad5 vector (rAd/E3/

TRE-Rep52) was generated by cloning the AAV-2 Rep52/40 coding

sequences downstream of the TRE and into the Ad5 E3 shuttle

vector pAB26 Recombinant virus was generated following

cotransfection of 293 cells with helper plasmid pFG173 and

pAB26-TRErep52/40 Various rAd and wild-type moi were used to infect

an optimal rAAV-2/Β-gal producing cell line termed D6 D6 has a

tripartite production plasmid stably integrated into the S1 locus and

contains: (i) a rAAV/CMV/Β-gal vector, (ii) p5-rep-cap, and (iii)

quantitated by visual counting of lacZ expressing target C12 cells

Results Efficient Rep52 expression was documented by western

blot analysis following infection of 293 or HeLa cells with rAd/

Rep52 in the absence of a tet transactivator protein To determine

infected with rAd/Rep52 at various moi (1, 10, or 100) Compared

to wild-type adenovirus infection, the recombinant virus yielded a

3-4 log reduction in infectious vector yield Under these infection

conditions, Rep western blot analysis revealed elevated Rep52 levels

that correlated with a dramatic reduction in Rep78/68 expression

levels A series of temporal coinfection experiments were then

performed by varying the order of wild-type and recombinant

adenovirus infection Using an optimal coinfection scheme (initial

wild-type Ad5 infection, followed 12 hr later by rAd/Rep52),

infectious rAAV/Β-gal yields were augmented approximately 8-fold

compared to wild-type Ad5 infection alone Moreover, under these

permissive infection conditions, Rep78/68 proteins were moderately

expressed Conclusions We report the generation of an E3 deleted

rAd vector expressing the AAV-2 Rep52 protein and define the optimal infection parameters for the synthesis of highly infectious virus from coinfected producer cell lines These data also suggest that overexpression of Rep52 helicase prior to Rep78/68 protein expression results in inhibition of subsequent Rep78/68 expression

106 Optimal Transductions of Mouse Embryonic Stem Cells with Recombinant AAV Serotypes (1-5)

Fusheng Wei,1 Sihong Song,2 Pedro Cruz,3 Yuanqing Lu,2 Jingfang Wei,1 Ge Zhao,2 Terence R Flotte.1

1 Pediatrics, University of Florida College of Medicine, Gainesville, FL, United States; 2 Molecular Genetics &

Microbiology & Pharmaceutics, University of Florida College of Medicine, Gainesville, FL, United States; 3 Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville,

FL, United States.

Pluripotent embryonic stem (ES) cells are derived from normal preimplantation blastocyst embryos In vitro, they can be stably propagated in the undifferentiated state, and retain their ability to develop into all cell types in vivo Pluripotent mouse embryonic stem cells may provide an effective means to analyze gene expression and function during development, and may provide a cell platform for the purpose of gene therapy Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that is capable of establishing persistent infections in a wide range of mammalian cell types Recombinant AAV (rAAV) vectors have been extensively employed

to mediate efficient gene transfer in the a wide range of cell types in vivo and in vitro There are at least eight serotypes, AAV 1 to 8 Vectors derived from AAV serotype 2 are currently being evaluated

in gene transfer studies To compare how other rAAV serotypes (1,3,4 and 5) perform as gene transfer vectors in embryonic stem cells would be particularly useful given their pluripotent nature

In the current study, ES cells were infected with rAAV serotypes

1 to 5 vectors designated to express human alpha-1 antitrypsin (AAT) at an M.O.I of 1,000 The levels of human AAT were measured by enzyme-linked immunoassay (ELISA) at 2,4 and 6 days post-infection The results showed that ES cells transduced with rAAV2, expressed hAAT at levels approximately 9-fold higher than other serotypes tested In another experiment, ES cells were infected with rAAV serotype 2 vector expressing green florescent protein (GFP) and the neo® gene at an M.O.I of 1,000 Cells were selected with G418 By 28 days, all G418-resistant ES cells transduced with the rAAV-GFP expressed GFP as judged by fluorescent microscopy Analysis of Southern blots of transduced clones demonstrated that stable vector integration had occurred To our knowledge, this is the first report of efficient transduction of mouse embryonic cells with rAAV Of particular interest is the possibility that sustained targeted expression may be used in embryonic stem cell based gene therapy

107 Isolation and Characterization of the Heparin Binding Domain of AAV

Yu-Hung Kuo,1 Justin F Fraser,1 Michael G Kaplitt.1

1 Neurological Surgery, New York Hospital, Weill-Cornell Medical Center, New York, NY.

While the various serotypes of adeno-associated virus (AAV) are believed to have the same physical structure, differences have been seen in viral tropism One possible mechanism for tropism is through specificity of the virus for different receptors The observation that AAV2 and AAV3 are capable of binding to heparin, whereas AAV4 and AAV5 show affinity for sialic acid supports this hypothesis Furthermore, it is likely that receptor affinity is encoded within the primary sequence of the surface capsid proteins Isolation and

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

BIOLOGY OF TRANSDUCTION characterization of the surface epitopes involved in receptor

association and selectivity would permit for design of novel AAV

forms with restricted or expanded tissue tropism

To isolate the heparin-binding site of AAV, we created chimeric

AAV isoforms where capsid domains of the heparin binding AAV2

serotype were substituted for equivalent domains in the non-heparin

binding serotypes AAV1 and AAV5 By using overlapping PCR to

create these chimeric viruses, no exogenous amino acids were

introduced Chimeric isoforms between AAV2 and AAV5

demonstrated that heparin binding was located within the VP3

portion of the capsid protein Replacement of the VP1 and VP2

sequence of AAV2 with the equivalent domain of AAV5 resulted in

production of functional viral particles capable of infecting HEK293

cells in vitro This AAV5/2 chimera was able to be retained and

purified on a heparin column In contrast, replacement of the VP1

and VP2 domain of AAV5 with the same domain from AAV2

produced infectious particles that were not retained by a heparin

column.To further delineate the minimal heparin binding domain,

chimeric viruses were made between AAV1 and AAV2, which show

more homology than AAV2 and AAV5 Substitution of fragments of

the C-terminal portion of AAV2 VP3 into AAV1 produced chimeric

virus that could be retained and purified on a heparin column The

chimera is infectious when tested on HEK293 cells in vitro The

heparin binding domain of AAV2 is in the C-terminal portion of the

VP3 sequence that is common to all subunits of the viral capsid It

includes the clusters of divergence between the amino acid sequences

of AAV1 and AAV2 Substitution of this domain of AAV2 with the

equivalent peptides of AAV1 ablated heparin binding Further

subdivision of this domain did not confer heparin binding, but appears

to adversely affect virus function We are currently determining

whether this domain is sufficient for conferring heparin binding to

other serotypes of AAV which are less homologous than AAV1 and

AAV2, as well as identifying the individual amino acids necessary

for heparin binding Finally, the effects of heparin association on

viral function in the brain in vivo are being delineated.

108 A Tropism Modified AAV2-Based Vector

System for Gene Therapy of Peritoneal Ovarian

Carcinoma

Wenfang Shi,2 Gregory Arnold,1 Jeffrey S Bartlett.1,2

1 Center for Gene Therapy, Children’s Research Institute,

Columbus, OH; 2 Department of Pediatrics, The Ohio State

University, Columbus, OH.

Ovarian cancer is one of the more common malignancies affecting

women in the United States Our goal has been to develop a

gene-based therapy for ovarian cancer using recombinant adeno-associated

virus (AAV) as a vector However, the utility of AAV vectors for

ovarian cancer gene therapy is limited by the distribution of heparan

sulfate proteoglycan (HSPG), the virus’s primary attachment

receptor, on different tumor cell populations In order to achieve

HSPG-independent gene delivery, several groups have shown that

the tropism of AAV can be expanded by genetically altering the viral

capsid However, the parameters of this developing technology have

yet to be defined and it has not yet been determined if these modified

vectors actually infect cells via these engineered interactions

We have analyzed tumor tissue from 40 patients with peritoneal

ovarian carcinoma for expression of HSPG and other receptors We

found that only about 63% of human tumors express the appropriate

HSPG epitope required for AAV infection, whereas greater than

panel of 6 different ovarian cancer cell lines (PA-1, OV4,

OVCAR-3, Hey, OV3 and SKOV-3) and similarly found that only half of

these cell lines expressed HSPG, yet all expressed either αvβ3 or

αvβ5 integrins Not surprisingly, those cell lines that failed to express

HSPG were not transduced with AAV2-based vectors

We have shown that incorporation of an Arg-Gly-Asp (RGD) – containing peptide at sites within the AAV capsid enables vectors

to infect integrin-expressing cells independent of HSPG Mutant AAV vectors displaying this peptide ligand were produced to wild-type titer and shown to specifically target integrin receptors on ovarian cancer cells Furthermore, we show that these modified vectors actually mediate infection via this engineered interaction

We report significant increases in gene transfer to previously non-permissive ovarian cancer cell lines, suggesting that AAV vectors displaying RGD peptides may be of utility for treatment of neoplasms characterized by a deficiency of HSPG expression We next tested these vectors for efficient transduction of ovarian

carcinoma in vivo Tumors were established by intraperitoneal

injection of SKOV-3 cells in SCID mice Either RGD-AAV2 vector

or unmodified AAV2 vector expressing GFP or LacZ marker genes was administered to the peritoneal cavity five days after tumors were established Animals were sacrificed at various times post treatment and gene delivery and expression were evaluated by immunostaining and enzymatic activity Both assays showed that marker gene transfer and expression was nearly two-orders of magnitude greater in RGD-AAV treated animals than in animals treated with unmodified AAV vector Importantly, the modified AAV vectors did not transduce normal mesothelial tissue or other internal organs RGD-modified AAV2 vectors encoding HSV-TK

have been developed and tested in vitro and in vivo Incorporation

of the RGD tumor-targeting motif in these vectors significantly

increased anti-tumor toxicity in vitro and efficacy in vivo These

results suggest that RGD-modified AAV vectors may offer significant therapeutic advantages for ovarian cancer gene therapy

109 Characterization of Adeno-Associated Virus Type 2 Minimal Origin of Replication and Packaging Signal

Sergei Musatov,1 Hajira Begum,2 Jill Roberts,2 Michael G Kaplitt.1,2

1 Laboratory of Neurobiology and Behavior, The Rockefeller Uniersity, New York, NY, United States; 2 Laboratory of Molecular Neurosurgery, Weill Medical College of Cornell University, New York, NY, United States.

Adeno-associated virus (AAV) inverted terminal repeats (ITRs)

are the only cis-elements required for virus propagation The 20-bp

D-sequence of the ITR has been demonstrated to play a critical role

in the efficient rescue, replication and encapsidation of the AAV genome While there is little homology between distal parts of the D-sequences of different AAV serotypes, all of them form a nearly perfect palindrome with the juxtaposed A-sequence This palindrome

generates a hairpin with a terminal resolution site (trs) in a loop

region, an event crucial for Rep-mediated endonuclease activity These observations lend support to the hypothesis that it is the secondary structure of the AD junction, rather than the primary sequence of the D-element, which is important for AAV propagation

To address this question, we first generated a series of circular AAV (cAAV) vectors with altered AD junctions Plasmids harboring a single wt AD domain have been shown to replicate exclusively as circular molecules and encapsidate single-stranded progeny genomes Vectors containing a single ITR element (AD domain) with no hairpin can be useful for mutational analysis of the D-sequence, since the mutant genomes can be generated using a simple single-step

PCR-based cloning All plasmids retain wt trs but differ in the flanking

sequences Southern blot analysis of replication revealed that the

plasmid with a GC-rich trs stem did not replicate efficiently, while

replication of a construct with an AT-rich junction was readily detectable Most importantly, generation of a single mismatch in the synthetic AT-rich stem increased the efficacy of replication to virtually wild-type levels Furthermore, we observed a direct