75 targeting of angiogenic endothelial cells using a new AAV 2 insertion site

75 Targeting of Angiogenic Endothelial Cells Using a New AAV 2 Insertion Site liver targeted approach,bloodcellscontainaround0 0I copiesofRV per cell at 5 years after transduction This low level hemat[.]

liver-targeted approach,bloodcells containaround 0.0I copiesofRV

per cell at 5 years after transduction Thislow-levelhematopoietic

marking and expression may prove to be an important component

ofthe therapy However, hematopoieticcell transduction also

pres-ents an additionalrisk for insertional mutagenesis in a population

of rapidly dividing cells As part of a long-term safety assessment,

blood from RV-treated MPSVII dogs that received direct injections

ofRV 4 to 5.5 years previously was analyzed for RVinsertion sites

using ligation amplification-mediated(LAM) PCR There were

relatively few insertionsites detected for each dog A total ofeleven

non-artifactualgenomic sequenceswereclonedfrom fivedogs The

results of a BLAT search of the UCSC May Z005 Dog Assembly

(http://genome.ucsc.edu/cgi-bin/hgBlat)allowed assignmentofnine

of these sequences to specific locations in the dog genome Of the

genes closest to the nine identifiedsites, themouse homolog offive

of'thesewere presentin theRetroviralTaggedCancerGene Database

(RTCGD(mm8) http://rtcgd.abcc.ncifcr[gov/) Assuming similar

genestructures in dog as in human,the locations withrespectto

genescould bedetermined, Twowerelocatedwithin 5 kb upstream

ofthe first known exon,three were within intron I, two were within

otherintrons,one was 14 kb downstream of the nearest gene,and

the last was at least 60 kb from the nearest annotated gene Three

of these dogs had also been analyzed three years previously In

that analysis,six unique sequences were identified Two of these

corresponded to genes in the RTCGD Only one site, within the

last exon of the HMGA2 gene,was detected in both analyses of

one of the dogs

73 Identification of Genetic Insulator

Elements for Safe Gene Therapy Viral Integrating

Vectors

CecileBauche,'Armelle Gaussin,'Julien De Royer,'Jean

Fran-cois Mouscadet,' Christian Auclair; Nicholas Merrnod,'Odile Y

Cohcn-Hagucnaucr,'-'

ILaboratoire de Biotechnologie et de Pharmacologie

Genet-ique ApplGenet-iquees, Ecole Normale Superieure, Cachan, France ;

21nstitute ofBiotechnology , University ofLausanne, Lausanne ,

Switzerland; J Department ofMedical Oncology, Hopital

Saint-Louis , Paris , France

Insertional mutagenesis has been demonstrated following the

integrationofgene transfervectors that includestrong

enhancer/pro-moters Conversely, the insulator approach can also be investigated

as a way to protectthe endogenousgenomicsequences/environment

from the risk ofdysregulation resulting from integrating vectors In

addition, recent reports have shown that genetic insulatorscurrently

under usc, like the HS4 1,2kp fragment or the 250 bp core clements

cannot wholly insulate the genetic environment from potent viral

regulatoryelements.In this event,there will be no garantecthat

side-effects will not reproduce followingclonal selection and expansion,

In order to identify short genetic elements capable of insulating

the strongest viral regulatory elements from the Fr-MuLV FB29

strain enhancer which are capable of both preventinginsertional

mutagenesis and the exctinction of transgene expression we have

established a standard screening procedure This assay consists of

a series of plasmids containing two reporter genes: one mimicking

a therapeutic gene under thc control of strong viral

enhancer/pro-moter, and the other one standing for an endogenous gene close to

the chromosomalvector integration site,Potentialinsulatorelements

interposed between the viral enhancer and the "chromosomal" gene

promoter are expected to shield the lattergene from the influenceof

the neighboringvectorelements.The setting up and assay validation

make use of the chicken beta-globin5'HS4elements, whereas

re-peats of short genetic elements having more potent insulatoractivity

are being screened and retained.New insulators are challengedwith

both the full FOCHA-LrR and the Fr-MuLVenhancer alone

Self-S30

inactivatinggammaretrovirus-basedvectors have been designed by other groups; the virus titers we have obtained from SIN Fr-MuLV FB29 derived,FOCHAvector,i.e.,106pfu/ml (Cohen-Haguenauer, unpublisheddata)are notstandard.Aseries ofinsulated gamma- and lenti- SIN-retrovirus constructs have thus been engineered using two combinations of the shortest active stretches, respectivelyof

268 bp and 157 bp long in total, substituting the U3 LTRenhancer

in full Thelenti backbone is derived from LuigiNaldini's

pSIN-18 (with kind permission).Data form these vectors and related comparisons will be presented In adddition, we aim at combining the usc of insulated lentivectors and targeting their integration to heterochromatin.Acknowledgement: EC FP6-NoE CLiNIGENE LSHB-CT-Z006-0 18933 (the European network of excellence for the advancement of clinical gene transfer and therapy)

74 Making Retroviral Gene Therapy Safer: Prevention of Retrovirus-Mediated Activation

of Cellular Genes near the Integration Site by Engineering of the LTR

YoungtaeHong,INam-KyungYoon,ISujeongKim,'Sunyoung Kim.!Joong Gon Kim,' JungWoo Rhim,'Hyoung Jin Kang,' Karim Lee,' Jiwon Jang.?

I Department ofResearch and Development Viro1l4ed, Seoul, Republic ofKorea; 2Department ofBiological Sciences, Seoul National University, Seoul, Republic ofKorea; 'Depanment of Pediatrics Seoul National University Hospital, Seoul, Republic ofKorea.

The usc of retroviraI vectors has recently demonstrated its actual clinicalbenefitin a few inherited diseases However,the leukemia cases foundafter thex-scmgene therapy trial has raised the safety concern of the insertionalmutagenesis inherentto the biology of the retrovirus.Although the retrovirus has long been known to integrate into the host chromosome,and thus have the potential to activate the nearbygene, there has been no convenient method of studying

or assaying such a cis-activation phenomenon.Herewe report an

in vitro assay system in which the effect ofretroviraI integration on the expression of the neighboring gene can be studied Using this assay, we found that the full-length LTRcould indeed activate the neighboring gene expression from a distance and the magnitude

of its activation was highly increased when this LTR was placed

in the vicinity of the transcription start site of the gene,while the truncated LTR exerted little influence.This system might provide

a useful tool for selecting the appropriate vector structure as well

as studying the molecular mechanism underlying the cis-activation

by the viral LTR

75 Targeting of Angiogenic Endothelial Cells Using a New AAV-2 Insertion Site

Jorge Boucas,' Kerstin Lux; Anke Huber,' Sibille Hummc.P MichaelHallek.t-' LucaPerabo,'HildegardBuning.l-'

'Clinic Ifor Internal Medicine University ofCologne Cologne Germany; 2Centerfor Molecular Medicine Cologne University of Cologne, Cologne , Germany.

The insertion of small peptides into the amino acid position 587

of theadcno-associatcd virus 2 (AAV-2) capsid opened the field

of AAVtargeting Since then, recombinant AAV (rAAV) targeting vectors with 587 insertions have been proven to yield increased transduction efficiency in vitro and in vivo and to transduce their target cell via the newinsertion.receptorinteraction.Furthermore, combinatorial approaches using AAV display libraries and high throughput-screenings simplified the creation of targeting vectors with desired transduction abilities In this report we demonstrate

Molecular Therapy Volume15 Supplement I• \by 2IlQ 7 Cop)Tight © The Amcricm Society o r Gene Ther apy

how,by analysisof the now availableAAV-2atomic structure, anew

position was found and improved to become a valid alternative to

587 By inserting a known peptide - RGD4C - into this new site we

were able to show a 3-fold increase in receptor binding capability

when compared to the same insertion at position 587 Increased

transduction was observed for target cells ase.g,primary angiogenic

human umbilical vein endothelial cells (HUVEC) Decreased

trans-duction efficiency for non-target cells was observed when

combin-ing insertions with mutationsof residues responsible for primary

receptor binding Specificity ofinsertion.receptorinteraction was

also proven by competition studies using soluble peptides

Further-more, a detailed analysis on different combination of insertion sites,

peptides, further capsid modificationsand target cells, allowed us to

define optimal transduction conditions and efficiencies According

to the analysis of the 3D-structure, peptides inserted at the new site

arc less prone to interfere with other viral functions required for

ef-ficientcell-transduction.By leaving R585 and R588 (main residues

responsible for AAV2's primary receptor binding) untouched the

new insertion site can be used to broaden AAV2's tropism On the

other hand,it can be used to redirect AAV2's tropism by combining

peptide insertionwith R585Aand R588Amutationsablatingprimary

receptorbinding (heparansulfate proteoglyean, HSPG) Maintaining

HSPG binding still allows purification and concentration of such

AAV targeting vectors by heparin affinity chromatography, and is

especially useful for ex vivo applications.Abolishing this binding

is of particular use for systemic applications since HSPGdependent

retention in liver and spleen can be avoided, thus increasing the in

vivo targeting ability of the respective vectors In summary, these

results expand the spectrum of targeting technologies for AAV-2

based vectors andoffernovel alternatives to address human gene

transfer related issues

76 In Vivo Selection of Targeted Gene

Transfer Vectors from AAV Random Peptide

Display Libraries

Ying Ying,IMueller Oliver,lWaterkamp Daniel,' Kleinschmidt A

Juergen.'

ITumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg,

Germany; llnnere Medizin, Universitaet Heidelberg, Heidelberg,

Germany; 3H aematologie und Onkologie, Universitaetsklinikum,

Frelburg, Germany.

promis-ing approach to increase the efficiency and safety of gene transfer

Our lab has successfully introduced a three-step protocol to produce

AAV-2 random peptide display libraries that ensure the encoding

ofdisplayed pcptidcsby the packaged AAV genome (Mueller et a!

Nat, Biotechnol 2003; 21: 1040-6).Selection of vector targeting

sequences on a panel of different cell types demonstrated that the

selected targetingsequences not only improved the efficiency but

also the specificity ofgene transduction.Our current study is aimed

at selecting targeting vectors which overcome the physical barriers

to the target tissue by in vivo application of AAV peptide display

libraries WeselectedAAV2 vectors for mouse cardiac gene transfer

by in vitro application on primary neonatal ratscardiomyocytesand

furthermore by in vivo homing to mouse heart after intravenous

injection ofthe displayed libraries Amplification oflibrary viruses

internalized within mice hearts was achieved by organo-typie

cul-ture of heart tissue slices super infected with Ad5 Specific peptide

sequences could be enriched after 4 rounds of in vitro screening on

primarycardiomyocytesor 3 rounds of in vivo biopanning in mice

hearts Selected mutants were then evaluated in vivo via tail-vein

injection into mice Analysis of gene transfer specificity and

trans-duction efficiency using selected clones will be presented

Molecular Therapy Volume15.Supplem ent I ~bl' 2007

C;oppight © T be American Soc i ety of Gen e Therapy

77 Genetic Modification of AAV1 Capsid for

MN Targeted Gene Delivery

Thais Federici,IAdam Davis,lQingshan Tcng,IJonathan Riley,I

Jeffrey S Bartlett.' Nicholas M Boulis.'

'Neurosciences , Cleveland Clinic, Cleveland, OH; lGene Therapy Center; ChildrensResearch Institute and Department0/ Pediat-rics, Ohio State University , Columbus, OH.

Background: Gene delivery to motor neurons (MN) using adeno-associated virus (AAV) vectors is limited by inefficient vec-tor binding to axons and the high affinity of AAV for muscle We proposed to genetically modify AAV to increase the transduction ofMNs by mimicking the uptake and retrograde delivery of tetanus toxin The binding domain of tetanus toxin (TIC), which binds trisialogangliosides (GTI b), was used to isolate a 12-mer linear peptide, Tetl, by eluting a phage library from GTlb with TIC We have previously shown that Tetl binds selectively to differentiated PCI2 cells,primary MNs, and dorsal root ganglia (ORG) in vitro,

and have performed competition ELISAexperimentsto demonstrate TetI-specific binding to the targeted GT!b receptor We have also

shown Tetl uptake at MN axon termini and retrograde delivery in

vitrousing Campenot chambers, as well as neuronal binding and

spinal cord uptake ofTetl after peripheral administration in vivo.

into the AAVI capsid may improve MV-mediated gene delivery

to MNs Methods: Our plan was to insert theTetl peptide into the AAVVI' I, VP2,and VP3 capsid proteinsby PCR-basedsite-directed mutagenesis ofthe AAVI Cap gene A site was chosen following

VI'I amino acid 590 based on our previous studies showing that this region of the capsid protein can tolerate small peptide insertions Packaging plasmids encoding modified AAVl capsid genes were used to produce AAVI.TetI vectors that were compared to unmodi-fiedAAVI vectors for their ability to transduce SH-SY5Y cells and for uptake at axon termini The molecular structure ofthe predicted

Tetl-modified AAVI capsid was also modeled in silico Results:

ModifiedAAVI vectors were produced and shown to increase trans-duction ofSH·SY5Y cells relativeto unmodified particles.Uptake

of vectors with modified capsid at axon termini was also markedly enhanced compared to the wild-type vector.Current experiments are underway to confirm the presence of the Tetl epitope in the capsid

of the modified vectors Although all evidence suggested that the TetI peptide was present and exposed on the surface of modified vector particles, structural modeling predicted suboptimal display

of the epitope This finding opens the possibility that efficiency and specificity mightbe further improved by additional modifications

to promote epitope display away from the AAV capsid Conclu-sions: These findings suggest that modifiedAAVvectors displaying neurotropic peptides might mediate enhanced gene delivery to the spinal cord Moreover, these experiments demonstrate our ability

to construct AAV vectors for MN-specific gene delivery through

genetic modification of the AAVCap gene.Furthermore, our mo-lecular model suggests that epitope display could be improved by subsequent modification and that even greater enhancement MN-specific gene transfer might be possible

78 Site Specific Modification of AAV Vector Particles with Biophysical Probes and Targeting Ligands Using Biotin Ligase

MatthewD.Staebler;' IrwinChen,'Alice Y.Ting,"Jeffrey S Bartlett.1,3

'Gene Therapy Center; ChildrensResearch Institute Columbus, 011; "Department of Chemistry; Massachusetts Institute ofTech-nology, Cambridge MA; JDepartment of Pediatrics, The Ohio State University Columbus Off.

Adeno-associated virus (AAV) has garnered considerable inter-est as a gene therapy vector due to its ability to transduce a wide

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