363 mouse models to assess the risk of vector insertional mutagenesis upon systemic delivery

363 Mouse Models To Assess the Risk of Vector Insertional Mutagenesis upon Systemic Delivery Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S141 R[.]

Molecular Therapy Volume 17, Supplement 1, May 2009

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 defi ne 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 infl ammatory signaling and together with inhibition of

complement activation by surface-bound fH account for the mild

infl ammatory 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

defi ne 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 infl ammatory and effector functions of complement are

inhibited by recruiting and binding serum fH

RNA Virus Vectors II: Vector Design and

Integration Properties

362 Cell-Specifi c 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 Scientifi c Institute, Milan, Italy; 2 FIRC Institute

of Molecular Oncology, Milan, Italy; 3 CUSSB, Vita-Salute San

Raffaele University, Milan, Italy; 4 CNR, Milan, Italy; 5 University

of Modena and Reggio Emilia, Modena, Italy; 6 National 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) fl anking

∼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 specifi c subsets of TFBSs

Analysis of sequences fl anking the integration sites of Moloney

leukemia virus (MLV)-derived vectors with modifi ed long terminal

repeats (LTRs), and of a human immunodefi ciency 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-specifi c transcription and

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-specifi c 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 fl anking 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 specifi c TFs or TF families

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; 2 San Raffaele University, Milan, Italy;

3 NCT, Heidelberg, Germany; 4 HSR, Milan, Italy.

Lentiviral vectors (LV) can transduce a broad spectrum of

non-dividing cells in vivo including hepatocytes Effi cient 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 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 infi ltrating 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 hepatospecifi c 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

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

S142

RNA VIRUS VECTORS II: VECTOR DESIGN AND INTEGRATION PROPERTIES

in a mouse model of liver-specifi c Pten defi ciency (14/22 treated vs

8/36 control mice, p=0.002), and in wild type mice in combination

with a tumor promoting CCl4 treatment (6/8 vs 0/9 in untransduced

treatment-matched mice, p=0.002) We are now exploiting these

validated mouse models to test the safety of therapeutically relevant

SIN LV Preliminary survival curves and HCC incidence readouts in

Cdkn2a -/- mice injected with these SIN LV do not show evidence of

genotoxicity Our approach will be instrumental to establish vector

designs with an appropriate biosafety profi le for liver gene therapy

Confers Effective Protection Against Insertional

Activation of Oncogenes in a Stringent

Tumorigenesis Model

Koyel Mitra,1 Jack Lenz.1

1 Genetics, Albert Einstein College of Medicine, Bronx, NY.

Tumorigenesis due to insertional activation of oncogenes by

transcriptional enhancers in vectors is a major problem in human

gene therapy Gammaretrovirus vector gene therapy for SCID-X1 has

resulted in T-lymphocyte tumors in several patients, marring what has

otherwise been a strikingly successful therapeutic advance Clonal

dominance caused by vector enhancer-mediated activation of EVI1

has also led to an adverse consequence Such problems are expected

to occur with any gene therapy vector that integrates throughout the

human genome and contains a strong transcriptional element, not

just with gammaretrovirus vectors Therefore the development of

new strategies to prevent enhancer-mediated activation of oncogene

promoters is a major need Insulator elements offer a theoretical

means for accomplishing this We developed a mouse retrovirus

lymphomagenesis model that rigorously tests the ability of the

insulator to block utilization of oncogenes on a genome-wide basis

and prevent tumors We initially found that one or two copies of

the chicken beta-globin HS4 (cHS4) insulator are not effective at

blocking tumorigenesis Therefore, we developed a new strategy of

using multiple, heterologous insulators in tandem The insulators

chosen were small and had different sequences, thereby conferring

stability during reverse transcription, a major issue because RT is a

jumping-prone polymerase We found that a tandem combination

of seven different elements (7X-Het) inserted near the 5’ end of the

LTR of a lymphomagenic MLV signifi cantly slowed development

of T-lymphomas in mice, although most eventually developed

lymphomas We now report the results of a genomics-based analysis

of oncogene utilization in tumor proviruses in those mice The

insulators were deleted from a fraction of proviruses, and these were

enriched among those adjacent to oncogenes In contrast, ‘insulated’

proviruses were enriched in proviruses near non-oncogenes ChIP

with quantitative PCR confi rmed binding of CTCF to insulators of

tumor proviruses When ‘insulated’ proviruses were situated adjacent

to oncogenes, they were usually oriented with the insulator near the

5’ end of the viral genome away from the oncogene promoter, one

occurring near the Lmo2 gene, a frequent culprit in the human gene

therapy associated leukemias Such orientations by design should

escape the insulators’ effect Only a small number of instances were

observed where a provirus containing an insulator was oriented

such that the insulator should have blocked utilization of the

oncogene Thus two uncommon events were frequently selected

in tumors of 7X-Het mice, deletion of the insulators or a provirus

escape orientation that evaded positioning the insulators between

viral enhancers and target oncogene promoters Most tumors that

occurred in 7X-Het virus infected mice were actually caused by these

uncommon molecular events that negated the effect of the insulator

Thus, the combinatorial insulator approach described here offered

much more effective protection against retroviral tumorigenesis than

originally realized, and it should signifi cantly improve the safety of

human gene therapy

Stem Cell Gene Therapy Model Using HIV1-Based Lentiviral Vector

Naoya Uchida,1 Kareem Washington,1 Matthew M Hsieh,1 Aylin

C Bonifacino,2 Allen E Krouse,2 Mark E Metzger,2 Robert E Donahue,1 John F Tisdale.1

1 MCHB, NHLBI, NIH, Bethesda, MD; 2 HB, NHLBI, NIH, Methesda, MD.

Although the successful application of gene transfer to hematopoietic stem cells for the hemoglobinopathies and thalassemias has recently been achieved in rodents using lentiviral vectors, preclinical testing in the rhesus macaque is hampered by a relative block to transduction by HIV1 vectors HIV1 vectors transduce rhesus blood cells poorly due

to a species specifi c block imposed by restricting factors, including TRIM5α which targets HIV1 capsid proteins (CAs) We sought

to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV1 and SIV, including SIV-CA and SIV-Vif We recently reported a chimeric HIV1 vector including SIV CA (χHIV) that can effi ciently tranduce both human and rhesus progenitor cells in vitro (Blood,

112, 11, 2353a, 2008) Using our system, existing HIV1 based therapeutic vector plasmids can be utilized to produce χHIV vectors

by simply replacing HIV1-Gag/Pol plasmid with that of χHIV-Gag/ Pol To evaluate whether this χHIV vector can effi ciently transduce rhesus repopulating hematopoietic cells, we performed competitive repopulation experiments in two rhesus macaques in which half of the CD34+ cells were transduced with the χHIV vector encoding GFP

at MOI 50 and the other half with a standard HIV1 vector encoding YFP under the same conditions The χHIV vector showed superior transduction rates in vitro compared to those of the standard HIV1 vector (fi rst rhesus: 61.1±0.36% vs 28.0±0.40%, second rhesus: 48.6±1.02% vs 30.7±0.51%, respectively) In the fi rst rhesus, in vivo gene marking levels derived from the χHIV vector transduced cells slowly increased in all blood lineages and reached plateau levels

of 15-30% 2-3 months after transplantation (Figure) In contrast, in vivo gene marking levels derived from the HIV1 vector transduced cells remained low at levels of 1-5% (Figure) Results were similar in the second rhesus; the χHIV vector showed superior marking levels (7-15%) in all lineage cells compared to those of the HIV1 vector (0.5-2%) These data demonstrate that our chimeric χHIV vector can effi ciently transduce rhesus repopulating blood cells, with high level persistence of transgene expressing progeny In summary, we have developed an HIV1-based lentiviral vector system capable of effi cient transduction of both human and rhesus blood cells which, unlike SIV vectors, should allow comprehensive preclinical testing

of HIV1-based therapeutic vectors in the large animal model