607 read throughsplicing capture mechanism is the major determinant of enhancer genotoxicity of vectors with active LTRs

607 Read Through/Splicing Capture Mechanism Is the Major Determinant of Enhancer Genotoxicity of Vectors with Active LTRs Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American S[.]

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

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RNA VIRUS VECTORS

T cell reconstitution of treated patients demonstrated recovery of

nạve HSV-Tkneg T cells The newly reconstituted CD4+ nạve T cells

were almost entirely comprised by CD31+ recent thymic emigrants

Accordingly, CT scans documented an increase in thymic volume

and single joint T cell Receptor Excision Circle counts rose following

HSV-Tk cell add-backs Comparison with a cohort of patients

subject to T-cell replete HSCT further suggested a unique direct role

of HSV-Tk+ cells in promoting thymopoiesis Interestingly, serum

levels of IL-7 markedly rose after Tk-cell add-backs, suggesting that

the genetically manipulated T cells may mediate the release of this

stromal cytokine, in turn supporting the generation and maturation

of T cells Notably, in the absence of HSV-Tk cell engraftment, no

increase in IL-7 serum levels was observed and patients did non

achieve the immune reconstitution The newly generated HSV-Tkneg

T cells granted persistent immune competence against infectious

agents, which was not compromised in those patients in whom the

suicide gene was activated to control GvHD These data show that

the infusion of suicide gene-modifi ed T cells induces IL-7 release,

boosts the function of the adult thymus and prompts the recovery of a

polyclonal, fully competent, T cell repertoire A phase III clinical trial

(TK008 study) to assess the effi cacy of HSV-Tk+ cells in the context

of haploidentical HSCT for leukemia started in Italy, and is currently

expanding to multiple centers throughout Europe and US

RNA Virus Vectors

606 Assessing the Impact of Lentiviral Vector

Integration on Splicing of Cellular Genes at the

Genome-Wide Level

Stefania Merella,1 Jacopo Sgualdino,1 Daniela Cesana,1 Fabrizio

Benedicenti,1 Simone Leo,2 Gianluigi Zanetti,2 Luigi Naldini,1

Eugenio Montini.1

1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy;

2 Center for Advanced Studies, Research, and Development in

Sardinia, Pula, Italy.

Oncogenesis induced by insertional mutagenesis with gene therapy

vectors occurs mainly by activation of proto-oncogenes found

at or nearby the insertion site This activation often occurs by an

enhancer-mediated mechanism or by a process of splicing capture

which generates chimeric transcripts comprising portions of vector

and cellular mRNAs Although the activation of oncogenes may be

reduced by the use of self-inactivating (SIN) design and moderate

cellular promoters, how to reduce genotoxic splicing capture events

and aberrant transcript formation triggered by vector integration is

still unclear We developed a modifi ed Linear Amplifi cation-Mediated

(LAM) PCR technique, named cDNA LAM PCR (cLAM-PCR),

aimed at retrieving, from the whole transcriptome of LV-transduced

cells aberrantly spliced mRNAs that contain lentiviral vector (LV)

sequences fused with cellular transcripts in a high-throughput fashion

The sequences of cLAM-PCR products were obtained by 454

pyrosequencing and analyzed by a purposely build high-throughput

computational pipeline Our pipeline is based on a map-reduce

parallelization model, running in a private computer cluster and use

a dynamic analysis process composed by different steps implemented

as map-reduce applications Thus, chimeric LV-genome sequences

are recognized, the nucleotide position of the fused sequence is

identifi ed (the splice site), and the remaining portion mapped on

the appropriate genome assembly by BLAST Results obtained

with different LV constructs show that integrated LVs can perturb

the processing of cellular transcripts by interacting with the cellular

splicing machinery and fusing with its own splice sites to cellular

splice sites both upstream and downstream the integration site So

far, 70 different fusion transcripts could be identifi ed in total, 84%

of which were fused to known splice sites of gene exons, 6% were

fused to uncharacterized cryptic splice sites located in introns and the

remaining 10% were fused to genomic sequences not corresponding to any annotated gene We identifi ed several established and previously unknown splice sites within the LV backbone that participate in the aberrant splicing process with variable effi ciency Quantitative PCR on different portions of the LV backbone allows measuring the relative contribution to the aberrant splicing process of each

LV splice site identifi ed The amount of transcription occurring in regions outside the expression cassette reaches up to the 3% of the entire transgene expression The cLAM-PCR technique, coupled to high-throughput sequencing and the computational power of our specialized data analysis pipeline allows gaining insights into the biology of vector-mediated splicing alteration Since this process could induce neoplastic transformation by the generation of aberrant oncogenic protein, its in-depth characterization is instrumental in the development of next-generation LV with a higher safety profi le

607 Read-Through/Splicing-Capture Mechanism Is the Major Determinant of Enhancer Genotoxicity of Vectors with Active LTRs

Daniela Cesana,1 Marco Ranzani,1 Cynthia Bartholomae,2 Monica Volpin,1 Stefania Merella,1 Fabrizio Benedicenti,1 Lucia Sergi Sergi,1 Christof von Kalle,2 Manfred Schmidt,2 Luigi Naldini,1

Eugenio Montini.1

1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy;

2 National Center for Tumor Diseases, Heidelberg, Germany.

We recently developed and validated a new in vivo genotoxicity assay based on systemic vector injection into newborn tumor-prone Cdkn2a-/- mice to address the genotoxic potential of different VSV-G pseudotyped lentiviral vectors (LV) Treatment with an LV with self-inactivating (SIN) Long Terminal Repeats (LTRs) and harboring the strong Spleen Focus Forming Virus (SF) enhancer/promoter sequences in internal position driving GFP expression (SIN.LV.SF GFP), caused a signifi cant acceleration (p<0.006) in the time of tumor onset (N=17, 186 days median survival) with respect to mock-controls (N=80, 233 d) These data confi rm the higher sensitivity to vector genotoxicity of this new in vivo platform since this vector was neutral

in the previously validated Cdkn2a-/- transduction/transplantation mouse model Injection of an LV containing the same SF sequences within the LTR (LV.SF.LTR) caused a signifi cantly earlier tumor onset even with respect to the SIN.LV.SF.GFP-treated mice (N=11,

138 d, p<0.0001) These results provide a further confi rmation that the position of the enhancer/promoter sequences has a strong impact

on the genotoxic potential of integrative vectors To understand the reasons at the base of the different genotoxic potential between vectors with the enhancer in internal position or within the LTR, we deleted from the SIN.LV.SF.GFP the Open Reading Frame (ORF) downstream the SF promoter (LV.SF) and tested the effect of this modifi cation in the new in vivo platform Strikingly, LV.SF induced

a dramatic acceleration in the time of tumor onset (N=9, 112 d;

Vs Mock p<0.0001) similar to the LV.SF.LTR This data indicate that the presence of the ORF downstream the enhancer/promoter sequences and not the position or the number of enhancers per se has

a major role in increasing the safety profi le of the SIN design To get more insights into the mechanism responsible for this difference in genotoxicity, we retrieved the vector integration sites from tumors Analysis of >5000 LV integrations showed the presence of several Common Insertion Sites in all the different vector treatment groups, such as the well known cancer genes Braf, Sfi 1, Mef2c and Map3k8 Thus, all genotoxic vectors tested promoted tumor onset acceleration

by insertional mutagenesis Furthermore, the LV.SF.LTR and the LV.SF produced with high frequency chimeric LV/Braf transcripts

by a read-through/splicing-capture mechanism using available splice donor signals present in the vector or in the surrounding cellular genome On the other hand, no LV/Braf transcripts could be detected

in SIN.LV.SF.GFP induced-tumor indicating that the presence of an

Molecular Therapy Volume 19, Supplement 1, May 2011

Copyright © The American Society of Gene & Cell Therapy S233

RNA VIRUS VECTORS

ORF reduces read-though transcription and splicing capture events

In conclusion, the major determinant of the reduced genotoxicity of

SIN vectors with respect to those with active LTR with a matched

enhancer type is their inability to generate promoter-driven aberrant

transcripts by the read-through/splicing-capture mechanism

608 Cell Surface Heparan Sulfate Proteoglycan

(HSPG) Is a Receptor for Foamy Virus

Md Nasimuzzaman,1 Derek A Persons.1

1 Hematology, St Jude Children’s Research Hospital, Memphis, TN.

Foamy viral vectors (FV) have several advantages over other

retroviral vectors for gene therapy They have a broad host range and

tissue tropism, the capacity to transmit a larger transgene cassette, a

DNA-based genome which is formed in developing virus particles,

and a distinct integration preference compared to other vectors

Despite the use of FV for over a decade, the cellular receptor for

the FV envelope has remained unknown Interestingly, we found

that FV transduction of human CD34+ cells was inhibited when

cells were prestimulated overnight on Retronectin-coated plates

but not when plated immediately on Retronectin-coated plates with

FV vector Since Retronectin has a heparin binding domain, the

possibility was raised that interactions with surface heparan sulfate

glycoprotein (HSGP) molecules on target cells might be inhibitory

FV transduction, was also inhibited in the presence of heparin

These observations led us to test whether HSPG is a receptor for

FV HeLa and 3T3 cells were treated with Heparinase III at various

concentrations to specifi cally digest heparan sulfate from target cell

surfaces The absence of HSPG on the treated cells was confi rmed by

staining with an anti-HSPG antibody Hepariase-III-treated cells were

10-20 times less susceptible to FV transduction than untreated control

cells In contrast, VSV-G lentiviral vector (LV) effi ciently transduced

both Heparinase III-treated and untreated cells (Fig 1.1) We next

treated four types of cells, HeLa, 3T3, Vero, and A549 with sodium

chlorate (a reversible inhibitor of proteoglycan sulfation) at various

concentrations The sodium chlorate-treated cells were signifi cantly

less susceptible to FV infection than the control untreated cells (Fig

1.2), while transduction with LV was unaffected

We also observed that two mutant CHO cells (but not parental

wild-type) lacking cell surface HSPG (Fig 2A) were resistant to

transduction with FV (Fig 2B), while LV vector showed similar

levels transduction in all three cell lines (Fig 2C)

Finally, we tested whether the cell line Raji, which both lacks HSPG and is resistant to FV transduction, could be made permissive through expression of HSPG We transfected a syndecan-1 (proteoglycan) expression plasmid into Raji cells and identifi ed multiple HSPG expressing clones All seven heparan sulfate expressing cell clones were rendered susceptible to FV transduction These results demonstrate that membrane-associated heparan sulfate proteoglycan serves as a FV receptor This fi nding may provide critical information

on the use of FV as a gene therapy vector

609 Gas6 Mediates Lentiviral Vector Entry through Apoptotic Mimicry by Bridging Virion Envelope Phosphatidylserine to Axl

Kouki Morizono,1,2 Yiming Xie,2,3 Tove Olafsen,4 Benhur Lee,3

Asim Dasgupta,3 Anna M Wu,4 Irvin S Y Chen.1,2,3

1 Division of Hematology and Oncology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA; 2 UCLA AIDS Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA; 3 Departments of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University

of California, Los Angeles, Los Angeles, CA; 4 Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, California NanoSystems Institute, University

of California, Los Angeles, Los Angeles, CA.

Entry of envelope viruses is typically initiated by binding of virally encoded envelope proteins to specifi c receptors on the cell surface Binding of lentiviral vectors is also mediated by viral envelope proteins, and their tropism is usually determined by the binding specifi city of envelope proteins that pseudotype the vectors

We have been developing targeting lentiviral vectors that can specifi cally transduce selected cell types Redirecting the tropism of lentiviral vectors requires both elimination of the original tropism

of the pseudotyping envelope proteins and conferring new binding specifi city of the selected molecules for the viral envelope We used modifi ed Sindbis virus envelope proteins to redirect the pseudotyped lentiviral vectors, and mutated the original receptor-binding regions of the Sindbis virus envelope protein Conjugation of targeting ligands successfully redirected the pseudotyped vectors Although we could achieve targeted transduction of many cell types in many experimental settings, we still observed non-specifi c infectivity of targeting vectors for certain cell types, including human primary endothelial cells This binding is mediated by a factor present in fetal calf serum that bridges the virus to target cells We attempted to identify the factor

by isolating it using ammonium sulfate precipitation and FPLC, followed by protein identifi cation by mass spectrometry We found that infectivity is conferred by bovine protein S in fetal calf serum,