National Library ($4 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services services bibliographiques

395 Wellington Street 395. rue Wellington Ottawa ON K t A ON4 Ottawa ON K I A O N 4 Canada Canada

The author has granted a non- L'auteur a accordé une Licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distnbute or seil reproduire, prêter, distribuer ou copies of ths thesis in rnicroform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

Tom Anastasios S tavropoulos Depünment of Microbiology and Imrnunoiogy

Submittrd in partial liilfillmenr of the requirements for the degree of

Master of Science

Faculty of Graduate Studies The University of Western Ontario

London. On tario. Canada November. 1999

O Tom Anastasios Stavropoulos. 1999

ABSTRACT

Helprr-de pendent herpes simplex virus vectors ( HSV amplicons show

considerable promise to provide for long rem transduced gene expression in most ccll

i> pes. Ttir current packaging system of choice for these vectors involves CO transfection

hith a hct of five overlapping cosmids that encode the full HSV-1 hrlper virus genornr

h m u hich rhe paçkiiging elements (pcic) have been deletrd. Althouph both rhe helper \,irus

and HSV amplicon c m replicate. only the latter is packaged inro infeçtious viral particles.

Sincc the titers obtiiined are too low for practiciil application. an enhünced second

seneration packaging system was developed by modifying both the hrlper virus and HSV

miplicon vector. The helper virus was reverse rngineered using the original f ivc çosmids to

gcneratc a single HSV-bacterial artificial chromosome (BAC) clone in E. coli. h m which

ihc p i c elciments were deleted [O jenerate a replication-proficient but pac kaging-de f tx t i ttt*

H S W Zenornc. The HSV amplicon was modifieci to contain the SV40 origin of

replication. which cicts l is an HSV-independrnr replicon to provide for the repliçative

c ~ p m i o n of the vrcror. The HSV amplicon is packagrd into infrctious paniclcs by co-

trnristèction with the HSV-BAC helper virus into the 193T ceIl line. and the resulting crll

Iyate is free of detectable helper virus contamination. The combination of b a h

modifictitions to the original packaging system affords an 8-fold increase in the yirld of

p x ka@ vrctor.

tieyvords: Herpes Simplex Virus Type- 1 (HSV- 1 ), Helper-Dependent. ,4rnplicon. Vrctor

Packaging. Bacterial Anificial Chromosome (BAC)

- - . I t l

ACKNOWLEDGMENTS

I wish to thank Marilyn iMcLeod for her excellent rechnical assistance throughout

i h i h projsct. and Xly Ciissam for providing the differentiated PClZ cells. Most imponantly

I wish to extend my appreciation to my mentor Dr. Craig A. Strathdee. whosr guidance

and wisdom hiis inspirited my passion for science and research. To rny parents and sistcrs.

I wcint to express my gratitude for their love and support throughout the course of my

siudies. Their sacrifices. for which I am cuntinually indebted. have rnabied me to pursue

itiy çareer goals with ecise. This work was supponed by an Ontario Graduate Scholarship.

TABLE OF CONTENTS

Certificate of Examination / ii

Abstract 1 i i i

.Açknouledgments 1 iv

Tablc of Contents 1 v

List of Tables 1 vi

List of Figures / vi i

List of Abbreviations 1 viii

Co-Authorship 1 x

Chüptsr I : Introduction

The Life Cycle of HSV 1 7

Virion Structure 1 5

HSV Gcnomic Oqanization 1 9

Regulation of Gene Expression in the Lytic Cycle 1 10

Viral DNA Replication and Packaging 1 16

Regulation of Gene Expression in the Latent Cycle 1 19

HSV- 1 Vectors 1 20

Hclper-Independent HSV Vectors 1 2 I

Hrlper-Dependrnt HSV Vectors 1 1 8

Objectives 1 37

Chlipter 2: An Enhanced Packaging System for Helper-Dependent HSV Vrctors 1 50

2.1 Introduction/51

2.1 mate rial and Methods 1 53

2.3 Results157

2.4 Discussion / 74

Rekrences 1 77

Cliaptrr 3: Discussion 1 8 1

Re terences 1 102

LIST OF TABLES

Table Page

I . I General charactenstics of HSV vectors 24

2.1 Enhancrd pxküging of an HSV amplicon vector 55

3.1 Cornparison of helper virus-free HSV amplicon packagine systems 87

LIST OF FIGURES

The Lifs Cycle of HSV- I

HSV- I Grnomc. Virion Structure. Lytic Cycle. and Origins

HSV- I Rcgulatory Cascade

Helper-inde pendent HSV- 1 Vcctors

Helper-Dependent HSV- 1 Vrctors

Recombinant HSV- 1 Constructs. Structure of the UL4 1-BAC Tÿrgeting Vector / The pHSvlac Amplicon Vector

Intsgrcition of the BAC Vector into the HSV- 1 ULJi Genr

Analysis of the Recombinant and Cloned HSV-1 Constructs by Field Inversion Gel Electrophoresis and Restriction Endonuclease Fingrrprinting

Transduced Reporter Genr Expression in Post-Mitotic Crlls

vii

LIST OF ABBREVIATIONS

bacterial artificial chromosome

blue Fonning units

blue foming units per millilitre

baby hamster kidney (thymidine kinase minus )

base pair (s)

Celsius (degree)

complementary droxyribonucleic acid

Dulbrcco's modified sasle medium

dimethy l suifoxide

deoxyribonucleic acid

double stranded

ethy lenediamine tetraacetic acid

hurnan imrnunodeficiency virus

kilobase pairs

mollir

milligrrtm

miIligram per millilitre

millilitre

microgram

microgram per microlitre

microlitre

millirnolar

moi

,Lnv

ng/ml

ORF

PBS

PCR

p t'u/ni 1

R X W A

multiplicity of infection

molecular weight

nanograms per millilitre

open reading kame

phosphate buffered saline

polymerax chain reaction

plaque forming units pet- millilitre

ribonuclease A

single stranded

simian virus 40

Tlienwphiltrs ciqricitictis

Tris EDTA

Tris EDTA !%Cl

Tris Boric acid EDTA

transducing units

Tris i hydroxymethyl) aminomethane

unique long

unique short

virus host shutoff

5-bromo-4-chloro-3-indol y I-b-D-gal actoside

CO-AUTHORSHIP

The following thesis contains material from a previously published manuscript co-

aüthored by Tom A. Stavropoulos and Dr. Craig A. Strathdee in the Journal of Virology.

. \ I I cxperimsntlil work was performed by Tom A. Stavropoulos and a version of the

original manuscript which appears in Chapter 2 of this thesis. was written by Tom A.

St~~~ropoulos and Craig A. Strathdee. For copyright relrase see Apprndiii.

CHAPTER 1: INTRODUCTION

Viruses are obligate intracrllular parasites that have evolved mechanims to

circiinivent the defense systems of their natural hosrs in order to replicate thrir srnomes and

ipread as non-living rntities. The specific strategies rmployed by viruses can range from

4mpk rscrptor protrins which mediate binding and tntry into the cellular cnvironmrnt. to

çoniplet inimunological evasive strategies that allow virus prrsistrncr throuy hout the

IiiCtinir. of the host. .Although viruses are notoriou~ly known for the harmful dl'rcth cliuwi

ii) thc i r hosta. most virus/host interactions in nature are qmbiotiç. The exchange oi'

nui r ien i \ . rritxibolic enzymes. shelter. and cvrn dmg resistancr. ciin provide a selestive

aduntase to a host when cornpeting for resourcrs in rhe scosystem. in its most simplit'ied

iorm the viruslhost relationships c m be classified as an exchange of information whiçh haa

c~o1vc.d ovcr millions of yrars.

hlolecular dissection of this interaction has shed light on viral pathogcnesis as uell

; is the cellular mrtabolic and immunological protective mechanisms of the host.

Historically. this information has been applied in the developrnent of vaccines or drug

ihrriipirs. Howcver. the field of senr therapy represrnts a different application which

c~ploith ihc ahility of the vinis to express its srnetic matririal in the hoht. Becausr of the

a d w u s made in the genrtic analysis of disrase. gene therapy hlis recei ved cnormous

public attention in recent years since it proposes a potential cure for many non-trecitablr

human disorders. Gene therapy clin broüdly be defined as the introduction of gcnrtiç

niiitrrial to the crlls of an individual to achieve a thrrapeutic benefit.

In order for this goal to be attained in the clinic. numerous technical and

phyhiologicai obstacles must be overcome. The most important of these are: i ) penetrating

host defensr mechanisms (delivery ) i i ) generating enouzh material for administration and

i i i associatins the correct therapeutic genes with a puticular disrase. Cntil thésc criteria iire

met. grne thcrapy wiil not be successful in most systems. The specitïc characteristics a

gene therapy vector must possess in terms of achieving this goal are: i ) efficient gene

iransfer and i i ) prolonged gene expression. In the p s t . physical methods of gsnr transfer

ti;i\ pi-ol.eri to be very inefficient i>i i1ir.o despitc their wide ipresll u w in cultiird

conditions. Therefore. bioiogicd agents have bren invesrigatrd as a more efficient

iippro~ich. suçh that the stringcnt requirements for genr therapy u n be fulfilled.

C~nfuriun;itrly no particulür vector system has been successful thus far. and despite the

c ihnb o t' rriciny researchers. gmrtic thcirapy remains a theoretical approach for the trcriirnr.nt

oI' hiirtiiin discase. Along with the adenovirus. adeno-associatrd virus. retroviruh and

Icntiviru*. the hrrpes simplex virus type 1 (HSV) is considerrd a promising biologiçnl

Jcliwry systcm. To successfully üchieve the goals set fonh by the scirntific çornrnunity for

gcnetic intervention in humtins using an HSV-büscd vector. î much s l r m r underst;inJing

01-rhe hililogy of the virus is nrtrded.

1.1 The Life Cycle of HSV

Herpes sirnplrn virus is a nwrotropic alpha herpesvirus that hüs a high prevalençr

ot' -SOCi w-opositivity (32. 43. 75 1. Hiimiins brcomr infectrd with HSV through intimate

contact with individuais that c a r y the virus and thus. HSV maintains its life cycle in the

ieneral population. The life cycle of HSV begins when epithelid cells of the skin or C

niucosal surface are invaded and utilized to produce progeny virions (Fig. 1 . I 1. From this

prima? site of infection. progeny vinons progressivrly spread to adjacent rpithelial crlls as

wll âs [O local innrrvating sensory neurons (95). Whrn a neuron is infected. the HSV

1.irion is transporteci via retrograde transport to the nucleus of the c r l l whrre the gcnomr.

ian ebtciblish a lifelong infection. In the mean tirne. a vigorous immune re'iponsr is

~timulatsd by the presence of viral antigens in the host. where eventually the symptorns

iissociatctd u. ith discase. prirnarily watery skin lesions. regress (95 1. h n y frrc virions Lire

diminatrd by nsutralizing antibodies of the humoral immune response. and an- host crlls

F I . I I . Thc lik cycle of HSV-1. Virus infects rpitheliiil cells ar a priniary 4 t t . of iii t k t i o n ( 5quarc.h i. and senerates progeny virions through the ;ictivntion of its ly tic cycle. \ Ï r ~ ( w [fien diwxnin;itr to adjacent cells and sensory neurons. where they traïrl bu rcinyi.ndc trmsport to neuronal nuclri and establish a latent cycle of infection. In latrnçy \ira1 po i i i t l s rcrnain quiescent and do not generiite any viral antigens until rracrivated. During rcnciivcition the virus first replicltes. travels back to prriphrrd tissues dong neuritcs h l ;in tci*>griidr. transport. and repl icates in epithelial cells causing a recurrent lesion ;it which point the virus c m gain entry in the CNS. ': Jhd i t ' i d t'rom Roizman (95).

Disseminated Entry to CNS

~rirnary es ion Infection Recurrent Lesion f l I

-

o e o o 0 0 0 .

Retrog rade

Establishment of Latency Reactivation

infcctrd by HSV are lysed through a cytotoxic cell-rnediated response (95 1. This

phenomrnon is characteristic of herprsvimses and is called the latent cycle of infection.

During Iatrncy. no viral antizens are produced and the genomes are indefinitel) maintaind

;ih iiiic1c;itcd lincar or circular DNA. For the herpes simplcx typr- I 1 iruh the predoniinatc

Gtc for thc maintenance of Iatrnr virus is the trigrminal ganglia of periphttral sensor)

nciiroiis (92. 1 I 1 ). Under normal physiological conditions HSV does not rrplicate during

I;~tcnq. ;~nd its gene expression protîlr is limited to at most a few genrh. Th13 chariicteristic

;illu~vs HSV to persist throughout the lifetime of the host in ii small population ut'neuroni

ii hich crrw as a reservoir for recurring infections. In some patients or during certain stress

conditions. for rxample hypothermia. axotomy. or ultraviolet light. latent HSV c m bc

ructiwteli from this state of dormancy (93 ) . Reactivation is the process where the viral

geiiomc is promoted to the lytic phase and new virions arc generatccl. Thr.3~ viruwr u-c

trun\portr.d bnck to peripherd tissues by anterognade transport. and Jisscminütion o f thc

i - i i - t i occiirb ro other epithdial çrlls and the central nenaus system ( Fig. 1 . 1 I. Evrntu:illy

ihc pnitiolo~ical cymptorns of disrasc return in the patient. and t h q iisuall! occur in thc

s m c tissues initially infected. The symptoms often Vary in severity depending on the

imniiinologicai susceptibiiity of the host ( 1 1 1 ) . The specific Factors conrrolline the

cstliblishrnent or reactivation of HSV in the latency cycle have not b e n clearly defined.

1.2 Virion Structure

The HSV virion is approximately 700-300 nm. and consists of four distinct

regions: i i ii dense core containine the lineu dsDNA grnome. i i ) an icoscidrltahedral protcin

capsid wroundin_o the genornr. i i i ) an amorphous tegument rq ion comibting of +cverril

L irion protrins. and i v ) an outer snrelope ( Fi j. 1-28 1. The prrsrncr of an arnorphous

region i tqument > bçtwren the rnvelope and the protrin capsid is a defining çharacvristic of

(il1 hcrprsviruses. This region is packrd with viral proteins which cire relrased into the

qtoplasrn whçn the rnvelope of the virus is fused with the cytopliismic membrane of the

cc11 during adsorption. Mernbers of this group includc: virion polypeptides VPI2. VP 16.

V P I 11 12. \.irion host 5hutoff (vhs i. the products of genes LTL9 and LrLIO and probably

orher prolrinl; noi y r t identitird (95) . The most common function of the tqument protein5

i b presumiibl y structurally related. howevrr. some mrmbers have s h o w rrgulatory

iunction. VP16 in pnrticular functions to up-regulate viral gene expression. I t x t s by

t.orniing a cornpleu with cellular transcriptional fiictors. prirnarily Oct 1. and rrcruitin~ rhe

cellular trlinscriptional rnachinery to the promoters of the first set of viral senes expressrd

(-37. 66. 7 1. 84. 57). This activation sets the stage for the lytic cycle. and signifiçcintly

conrrihutrlh to its efficiency.

In cimtrast. the vhs protrin increases rhe effiçiency of viral gene txprehhion h)

dcgr~iding the pre-existing pools of mRNAs in the cell. This function dlow\ viral rnwogrts

un i nipcdcd access to the triinslational rnachinery of the crll i 29 1. Furthermorc. v hs

acccnruares c hnngcs in viral genr expression because i t afso non-sprçificd 1) d e p d e s viral

niRNAs. although at ii lower efficiency (95). So fiir the functions of the other tesurnent

prritcinh hiive not been adequateiy delineated.

HSV can infect a wide spectmrn of mammalian cells. including most crll types in

thc humlin body. This ability is cmied out by the nurnerous surface glycoprotcins on the

virus cn\dope. They includr glycoprotein B (gB). 5 0 . gH. gK. and gL uhich are eswmal

tor \iru\ srowth. and six othrr non-essential glycoproteins (SC. SE. 2G. gI. 2J. and $1)

i Fig I . I B 1. Initial aitachment of HSV to susceptible cclls is mediated throuzh chargrd

intc.r;ic.tiun\ betwern gB and gC on the virus envelope. and ccll surface

glyxsiiminoglycans such as hrparan sulfate or chondroitin sulfiitri i 10. 49. 50. 72. 80.

102). .Adsorption or membrane penetration occurs in a two stcp procrss following

attlichmrnt < Fig. 1 .?CL First through specific interactions betwern gD on the virus and its

cellular rrceptors HveA or HveC (named for herpesvirus sntry mçdiarori (85. I I 4 . The

second 3tep involves fusion of the plasma membrane with the virus envelope. Although

FIG. i 2. A Linrar representation of the - 152 kb HSV- 1 genorne. Inverted repèats are ~cp:!r31~1J h i t w unique sequcnces (LIL and Us) and shown as boxes with thcir orientation Lic~c.~-ihcd h l the c. a. b / c ' . a'. b' notation. Origins. packaping signais. and grnes are 3s I I I ~ ~ C ; I I L ' ~ . B I Stru~ture 01. the HSV-1 virion. C) Cartoon representation of the lyric cycle oi HSV- 1 . I I .-\irachment of the virion to cellular recrptor HveA or HveC. 2 ) Fusion of the \ Irilh cnwlvpc. and thc cytoplrtsniic membrane of the cell 3) The DNA containing capsid ; i i d tcgurncnt protrin are relsasrd into the cytoplasm. 11 Transloçarion of the capsid and p < m c d o n g the çytoskrletril network of the ccll to nuclear pores 5 ) Cncoating of the i;ip\id ;ind DSA penetration of the nucleus 6) Rrcircularization of the linrlir srnome to a iircular configuriition 7) I f a neuronal cell is infected. the genorne can enter the latent cycle iind c5t;ihiish ;i persistent infection (no viral genes are expressrd) 8 ) The viral penoms is i-cacti i ~1tl.d and snters the lytic cycle. Progeny virions are producrd as a result of viral gene cspi-c\hion. 9 1 Translocation of VP 16 (tegument protein) ro the nucleus and transactivation ot t r 2CtlL.r IO 1 TI-anscription of the a genes 1 1 ) Translation of u mRNAs into proteins 12 > TI-iiii;locati«n ul' ICPJ. LCP27. ICPO to the nucleus and viral P grnr expression become ;ic.ti\ ;LICL[ 1 3 ) Transcription of P mRNAs II) Translation of the P messages which givr rise tu thc polypeptides involved in DNA replication. 15) P gene products replicatr the viral D N A A i Initiation - UL9 protein bind the viral origins and mclts the duplex with the aid of [CPS. B ) Elonyarion - viral or host replication machinery resolves the e structure gsnrrated ibl lin\ in2 initiation. C) Rolling circle replication - viral enzymes gtinerate long concatrmrrh ot' itiiiliipls penonit. units. 16) Switch in gene expression trom the P penes to the gent.\. I - 1 K P - I ;mi1 ICP27 tninsactiwtion induces the production of y rnRN.4~. 18 1 Trandation d' -,I tiicb\ageh which compose of the structural components of the virus. 19) Translocation of thc \tructurlil proteins to assembly centres at nucltiar pores. Cleüvage and packaging of indi\iduiil viral senomes into protein capsids. 20) Virion maturation whrre DNA containing capids cicquire a glycoprotrin coated envelope and a series of tegumrnt protrins. Mature \ irium rcmoin membrane associated in the rndoplasrnic reticulum and transportrd into the c.;trciccllular spacc. D) Structure of the HSV-1 origins of replication. Nucleotidrs i n bold iuiicatc sequsnce diffrrences between the oris and oriL. Arrows show the relative oricniation and location of the repeat recognition sites for the LrL9 origin binding protrin i Box I. I I . and 111 1. and the boned nucleotides represent the palindromic A+T rrgion.

ac LAT ----*--LAYCH - - SV- & I I A

Envelope Glycoproteins Tegument Proteins gB, gC, go. gE. gG, vhs, VPf 6, VP22,

gH. gl, gJ, gK, gL, gM VP11112, US9, US1 UL46, UL47

Icosahedral capsid Linear dsDNA genome

- - BOX III BOX i BOX i BOX III

Y

th15 niechanism has not bren elucidated. it is dependrnt on SB. D. and H function (25. 76.

99, Thebc two svrnts expose the viral DNA. which remains cnclosed in a protrin capsid.

io the interniil environment of the cell. During this process the viral tcgumrnt proteinj are

rclc~ised into the cytoplasm of the cell (Fig. 1 . ?Cl Once inside the cell. the viral capsid is

rr;insportcd dong the cytoskelrtal network of microtubulrs and uncoatcd at the nucleus i 2.

105). Prcçirely how uncoating of the viral DNA takrs place is not clcarly undrrstood.

houevcr. it is clear that some viral function is reqiiired. This is rvident because capsids of

ihc tcmpcrciture sensitive mutant tHFEM)rsB7 only rrlease the viral senorne into the

iiiiclcii\ ;iitr.r A h h i f t tu the permissive tomprraturc. FinaIl). the geiiunir. penr.triiir\ ihc

riuclsu5 of the céll. is rapidly recircularizrd frorn its linrar predecessor. and rntrtrs cithrtr the

latcni or Iytii. cycle of infection (95 ) .

1 .S HSV Genomic Organization

Genetiçnlly the virus is weil chüracterized. with ri doublc strandrd DSA grriorne of

;ippro.irini;itt.ly 152 kb (95) . The genome is organized in two stretchrs of unique seqiicnces.

dcnotcd il:. unique long tuL) and unique short (Us). each bracketed by invrrtcd repeats

i Fig. 1 . 3 >. Because the invened repeats rire diploid. a unusual phrnomrnon occurs to the

\) rnmetr! of the viral genome. The unique sequencrs C L and Cs. reciproçall) tlip rt.lüiiw

t i i c x h othcr. suçh that four distinct equimolar DNA isomers are packagcd in progen) virw

plirticlrs. This process is known as inversion and is thought to oçcur ris a result of

recornbinlition brtwrrn homologous sequences in the invertrd rrperits of the gsnornr.

Grnetic cinalysis of the 500 bp ci sequence regions of the repeats s u _ r p t t h t x rlenirnts are

important to this process since mutants with small deletions in thesr aretis show a drastic

rcduction in this frequency (3. 1. 52. 93). However. the precise mechanism or

phyiiolopiçiil function of this pmcess in HSV biology remains unknown.

Currently there are 89 recognized different open reading frames within the HSV

senome. Five of these are diploid because they are located in the invrrted repear rqion.

Ertch grne can be categonzed into one of three different classes as deîïned by their rrrnporril

expression during the lytic cycle: immediate early (a). early ( P) and late t 7 ) genes (241.

Thcrr: are only five u genes. uJ. a?. aO. 021 and u47 (951. Thrse genes are the first to be

espresscd in the lytic cycle. and the most important becausc thcy transcriptionally anci

ames. tiankttioniilly regulate the expression of the remaining P and 1 ,

1.1 Regulütion of Gene Expression in the Lytic Cycle

During thc Iytic cycle. viral triinscripts are prnerated i n threr. succcshivc wiivch

hcpniiing uith the ci triinsçripts. and thcn followed by P and -{ mRN.\s (Fig. I .:Ci. This

tcnipor:il progüm ensures the correct viral proteins are made at the appropriate timt.3. The

V P l h tepnient protein initiates this program by acting (LÏ ÿn u-gene speçific transaçtivator.

I i docs this by binding host transcriptional factors. and recruiting the cellular RSA

pi~lymcrasc apparatus to VPl6-responsivs slrmrnts in the promoters of al1 u yeneh 166. 7 1 .

$4. S7 1. Thrrefore. the lytic cycle is dependent on the transcriptional mxhinery of rhe host.

Transcription of the f ivc u genes peaks at 2-4 h postinfection. Following tr:inslatiim

t ) t rl~chc i i i c h s q e h into protein. the products: infrctrd-cell-polypeptide ICP4. ICP27. ICPO.

ICPE. and ICP-17. respectively. constitutr the first viral proteins made in the Iytic cycle.

mi proniutc the viral replication phase. Of the f ivr a protrins. only two ( ICP4 3rd ICP17i

are essent id for virus growth in cultured cclls. The others. ICPO. ICP22 and ICP47 are

diqwnsable. however thry significantly inçreasc the rfficiency of the Iytic cycle.

plirticuiarly i l i iivo (95).

Following the first stage of viral translation. 1CP-l. ICP17 and ICPO are

trrinsloçlitcd to the nucleus (Fig. 1.2C) (95 ) . These threr re_oulate the expression of the rest

o f the genorne by first acting as transilctivators of P grnr transcription- B!, recruiting the

R X A Pol11 complrx to the promoters of P genes and activating their expression. replication

uf the iiral genomr soon follows. Synthesis of B mRNAs succeeds rr eene translation by

iipprosinicitsly 2 hours and peaks 5-7 hours postinkction. messages ~ i v e rise to the

products necessary for viral DNA replication. including. a viral DNA polÿmerass.

iinslc-5rr;ind DY,\ binding protcin. origin binding protrin. and the hsliclisdprimli~r:

~ .on ip l c s . B \ l 5 hours postinfection most viral DNA synthesis is cornplrtrd.

At [hi'; point long herid-to-tail concrttsmeric units of HSV grnomes riccumulate in

the nucltiiis while the 7 grnes are transcribed. The -( senes are transxtivrited by the u

1-c~iilator) grnes and expressrd only aftrr the B gencs have replicatrd the yenonie. This

w i t ç h in gene expression coincides with a drastic drcrease in p gene expression and

~ipnals thc üssembly phase of the lytic cycle (95) .

Synthesis of y mRNAs psaks at 12- l5 hours postinfection. and çonstitutrs the last

group of polypeptides made in the lytic cycle. The majority of genes code for structural

coniponcnts of the virion. including the major capsid protein (ICPji . envclopc

gl!cuprotc.in\ igB through yH). tegumsnt proteins. as well as. the enzyme3 and wbunits

rc.quiscd to iis\emble these structural components into a mature virion (Fig. 1.X) ( 9 5 ) .

Tlicw primins cleavc the DNA çonçatrmcrs inro single srnome Irngth unitz. packqe the

gcnunies into protein capsids. i n t e p t e the trgurnent proteins wirh D U contriining

capsids. and surround the maturing virion with a glycoprotein coated envelopc (77.23.79.

1 12). Cleawpe or genome packaging is dependent on the cis sequences lociitrd in the

inw-tcd rcpeats. Wild type HSV has two of these elernents and they are referrrd to as

packqing or ptrc rlements (Fig. 1 .?A). Deletion of both the pclc elements from the gcnornr

i b required to librogate the ability of the virus to produce mature progttny virions ( I I ? ) ) .

Gra l gsnomes which do not contain any pcic elements. are thus rrfrrred to a b

pltck~i_jin~-defrctive. bloreover. recombinant viruses with only one poc eiement , mene rate

proptin? virus as efficirntly as other HSV viruses that have two. and this clrmrnt is full'

iùnçtional rrgardless of its position in the genome ( 16. IM).

Progeny virions remain membrane associated in the endoplasmic reticulum once

budding through the nuclear membrane occurs. and in as little as 16 houn post infection

the lrtic cycle is completed and virions spread to the neighbouring çells t Fiy. I .2C) (95 1.

ICP-I. iCP27 and ICPO regulate the precise changes in gene expression from u to fl to -/ b)

cimiplru transcriptional and translational mechiinisms discussed below.

ICP4: Stmcturally. ICP4 is a 132 kDa phosphoprotein that contains a high iiffinity

DN.4 hinding motif in the carboxyl half of the protein. The first binding site idrntificd was

ATCGTCnnnnCnGnn. although others have since been reponed which do not çonform to

[h i \ consensus i 30). These binding sequences cire prescnt in HSV promotcrs and art. cnicial

I O thc ir;iri\activlitin~ funcrions of ICP4 (83. 9-11. ICP-I is probiibly rhc n i w imporrcint

rcgi~laton protrin in the virus lytic cycle bçcauss i t affects the transcription of dl threr gene

c labx \ and i ls such. delineating its precise role in gene regulation ho?; been confusing at

t l i n C s .

Thc niost informative ifau cornes from studirs usin3 d nul1 n i u e n t h . Thfie r iruwb

do not replicm nor produce any progeny virus. such that phrnotypically . the? express only

ihc rcniainirig u srnrs 114.95). Therefore. ICP4 is belirvrd to regdate the expression of

and genes. which has now been confirmed in othrr biochemical studirs. Additionallv.

ICP-I nqlitively regulatrs u _orne expression. and controls the Iwrl of rrtinscriptim t'rom

cach virlil promotrr temporally (Fig. 1.3). It is unclear precisrly hou. 1CP-l accornplisheb

t h ir di\criminlition between the different class promoters. however. an inçreasin_o body of

hiwhr.micii1 data wggests that heterosrneity in the cis bindins sequences of promoters as

urll as posttrtinslationcil modifications to ICP-1 play importsnt roltts. For eurnple.

phosphorylatsd forms of ICP4 bind promoters of p and .{ ?enes. whereas non-

phospliorylüted forms of ICP4 bind promoters of a genes (86). Therefore. the vims can

iitilizr the modification of the affinity of ICPJ for different promoter sequences as a

transcriptional mrchanism to exert control of almost rvery viral gene in the senorne.

ICP27: ICP17 is the other essential a gene. It is a nuclear phosphoprotein that

bindh single-strandrd DNA. and is known to regulate viral senr expression t Fig. 1.3

FIG. I .3. The HSV- 1 reguliitory cascade. The expression of HSV- 1 a. P and y gcnrs tire scyl~iicd and Jependent on the functional a proteins ICPO. [CPY and [CPJ. The u genes i1CP-I. ICP27. ICPO. ICP22. and ICP47) are transcribed by the R N A polymrrüse I I Iiolociiz~~nic. O C the host following nuclear penetration of the viral DNA and V P l 6 miiulî t i tm. The DNA replication enzymes (P genes) transüctivüted by ICP4 and ICPO. I i u c rcplic;~~cd the grnome into covalrntly linked head-to-rail concütemrrs by 15 h powiicctiim. Swl> qnthesized genomrs are packaged into the structural componenrx of [tic \ irii in. ir hich arc rncodrd by the 7 genes. and inducrd by ICP4 and ICP27. genr iriinxriprion peaks 8- 15 h postinkction. and corresponds with a drcreasr in 13 gcnr cxprc~siuii. ir gene espression is regulüted by KP4. md prevrnts the synthrsis of nsw u p l ! pcptiilch 5 h postinfrçtion.

'. JIdi t ' i d t'rom Fink et cil. 1996 ( 3 2 ) .

i 1 I Y i. Its DXA binding characteristic is believed to mediatc the association of ICP-7 uith

tlic RY.A Pol11 cornplex during transcription. and thus provide the caralytic dornains of

ICP27 positional access to actively transcribed senes. The most informative evidcnce

riipporting this virw cornes from genetic studies using ICP27 nul1 mutants. Thrse murant

i.irusr.s do not replicats or produce üny progeny virus becausr of a Iaçk of iransiiciivÿtion of

ihc il 2enr.s in the lytic cycle. Even though ICP27 nuIl rnutitnts produce a maIl ~ b w i o i

u id -! pal' peptides. this level of expression is insufficienr for virus replication let alone

i i r i c i n pruduction (95) . Furthrrrnorc. additional studiçs sugsest that ICP27 is a

~iii~ltit'iinctic~~i~~l protein. and currently the charrictrristics attributd to i t Lire: i i I deterniineh

ir hich termination signal is used whsn more than one is present. i i i i inhibits R N A bplicinp

h~ wqiirstering snRNPs. and ( i i i ) destabilizes a mRNAs. restrriining their translation (-17.

9 1 . 9 2 ) . iC;hcthcr or not al1 these arise from the direct action ICP37 or to downstreani

wtcomcs of the regulatory cascade rernains unclelir.

ICPO: ICPO is third potent iictivator of viral sene expression. howrvrr it i h not

c ~ c i i t i d for rhc process. I t acts prior to the start of transcription and iip-re=uliiter the

cqxcb\iun of the fi and y viral p e s (Fis. 1.3) (9. 17. 28. 42. 961. Prcçisely huu rtimainb

unclex sincc ICPO does not bind iiny viral promoters. nor is the expression of any , uenes

dcpcndcnt on a functional protçin. Again the most informative evidencr has corne frorn

cspcrinients usin: ICPO nul1 mutants. These vinisrs have impaired growth kinrtics brçauss

ot' thcir Jelayrd expression of the P and -{ genes ( 14. 96. 1 13. 1241. This phrnot-pr is

fiirther accrntuated at low multiplicities of infection. Even though ICPO is not absolutrly

rcquired for growth. these mutant viruses are severely compromised and cannot effectiwly

sstablish disrase in animal models. Therefore. ICPO is considered an important cornponent

of rhr I!;ric q r l e and a significrint virulence factor.

ICP22: The a22 grnr encodrs the ICP1-2 protein. It also is not essenrial for virus

y x r t h in çultured cells but tippears to be an important virulence factor. Nul1 mutants of

a22 phenotypically show a delayed shutdown of P gene expression following class

s\i.itching to the -1 grncs. As it result these vimses have decreüsrd levrls of -! genr producth

ii hicti iii;il\r.$ thsm sionifiçantly less piithogenic in riva. In ceil culture h u w w r . ICP22

mutants iyically grow much slower. and generatr k w s r progeny virions per infrctious

c.~,cIt' 18. 17. 90. 95).

ICP47: Unlike ICP77. ICP47 is not involved i n viral sene replation. but also

contributes w the piithogenicity of HSV. Specifically. ICP-17 has lin ininiiinomoJularor~

î'iinction i ~ t vira since it interferes with the iibility of the ceIl to prrsent viral antigrns on irs

iiirtacr. I t acts by binding the TAP transporter molccules of the MHC class-I pathway.

w h that viral peptides generated by the proteosome are not rfficiently transportrd into the

Iiinicn O!' the endoplasmic reticulum (36. 38. 5 1. 119,. This fiinction a l l w i the \ iriis to 20

~iridctccicd by the host by rscripinp the cytoroxic T-lymphocyte responws of thc irnrnunc

r!rtciii ;mi dosb no[ affect peptide presentation by the MHC class-11 pathwiiy ( 4 4 .

Thcrefore. by prrventing immunologiçal recognition of virally infected ceils. HSV clin

csthlish n-iorc efficient infection i r t riw.

1.5 Viral DN.4 Replication and Packaging

The viral proteins rrquired for DNA replication and nucleic acid metabolism arc

cncoded by 13 grnes. Thrse genes cannot be transcribed unti l the u regulatory protcins

i 1CP-l. ICP27 and ICPO) iransactivate their expression when the host's RNA polIl

coniplsx is rrcruitrd to P gene promoters. P gene transcripts c m be detrctrd as carly as 3

hourh postinfeçrion. and their presrnce signais the DNA replication phase of the Iytic c'clc.

During this phase HSV genomes are rrplicatrd through a predominately rolling circlr

mechanim. such that. covalently linkrd çoncatemers of hrad-to-tail rrpeat units are

~enercited i 5 i . AH the enzymes as well as the cis sequences invoived in generating thesr

concatrmers are essential for virus growth (95).

Foilowing uncoating of the v i n 1 DNA from its capsid iit the nuclrar membrane. the

lincar gcnomr is quickly recircularized upon penetration of the nucleus. This structure is

hiniilai IO the replicating grnomes of bacteria because the? both maintain a circular

configuration. In a circular format the ends are structurally similar to the rest of the viral

DS.4. and thercfore. provides a convenient way to repiicatr the ends since on1 y one type of

replication apparatus is required. Uniike cukaryotic organisms which have linear

chroinosonies and require two replication complexes: i ) one to extcnd the replication fork

fdlowing initiation iit the origin and i i ) a speciaiized telornerase apparatus to maintain the

Icngrh of the tslomercs. HSV does not have to encode for additional enzyme3 involceci in

tcrmiri;il rcp1ic;ition.

Thc HSV genome contains thret: origins of replication which are essential for DNX

rsplic:iiion. I w o copies. named mis. are locatrd in the c and c' rqions of the invertecl

rcpcilis. and une copy in the L' region ( ()riL) 1 Fig. 1 .?A 1 (6. 95 i. Thesc origin are large

piilinclrorniç rcpeats rhat contain an 18 hp A+T rich @on in the centre fllinked bç

cimserwd cibs srquences (Fig I .ID). These cis sequences are designated Box I and Box III

mi contain invcrted repeats that serve as binding sites for the origin binding protein

cncoded hy UL9 (6. 16.46.48. 73). Association of the UL9 protein with the Bon regions

ot' thc origin sipnüls the organization of the single-stnind DNX binding protrin. ICPS. rrith

[h i \ cornplex (-16. 73). Both ICPS and the UL9 protrin arc rrquircd to initiate DL!

rcplicciiion in which the first step is the rnelting of the 18 bp A+T region of the orizin ( 7 4 .

Thc eners)- rcquired to melt the duplex DNA is provided by the ATPasr xtivity of the U L Y

pruiein. and its hrlicasc functions to drstroy the hydrogen bonds b t t w e r n ihti

çornplrmenting strltnds and release single-stranded DNA (74. 8 i ). The SSDNA sr= umrn ts

1ibrr;tted by the action of the UL9 protein are quickly bound in 12-23 nuclrotides portions

pcr ICPS moleculr. in a highly cooperative manner. in order to prevrnt their reannealins

(45 ). Stabilization of this dissociated duplex sîgnals the recruitrnent of the remaininp

subunits of the replication cornplex. and the genome is duplicated (78) .

This mrchanism of initiation governed by 1CPS and the LrLY protein strongly

suggests that replication proceeds through a 0 intermediate ( 5 ) . This is truc becausr

dissociation of the A+T region of the origin in a circular DNA molecule. yields diverging

replication forks that can be resolved into two intact genomes following slongiition. Despite

niiinerous efforts to reconstitute the 8 mode1 of HSV DNA replication. thesr formh hiive nor

hccn ohrcrvd iiz i i i w to date < 100). Thrrçfore. the importance of 0 replication to HSV i b

iiiconclusiw and has not bssn clearly defined since the majority of the newly synthrsizrd

w-d gcnonieb cire gcnrrated by a rolling circlr mechünism (61. Rrprdless. i t is widely

hclicwd thüt HSV replication first occurs throuzh a e intermediate. and (it wrne poinr a

witch o w r to rolling circle replication happens. whrre long head-to-tüil concatemers of

niiiltiplc senome units are producrd (5. 101).

The rolling circle mechanism used by HSV is believed to br: similar to replication

btrategy of lambda bacteriophages. It involves nicking of one strand of the double-strandcd

penonit. and displacement of the 5' end such thüt the 3' end is uscd as ü prinicr for

clmgitiun by the DNA polyrnrrase çomplrx ( 5 ) . The other strand rernliins in a circuliir

coniiyration and serves as the template for unintcrruptsd DKA synthesis whrrr long

conciitemers of multiple DNA copies are generated ( 5 . 101 ). The complrmrnta~ stründ for

the displaced ssDNA is subsequently made in srnall segments using RNA oligonucleotides

ils prinicrs for extension which are generated by the primase activity of the polyirriisc

huloenz)~mt.. Second strand synthesis procerds in a mannrr typical of la=ginr strand

y t h e s i s where the RNA nucleotides are removed and the Okazaki fragments ligatrd

togerhcr to form an intact DNA strand (5. 101).

The structural intermediates generated by ro l l iq circle replication haw bsrn

u b i m u i i i i i i r - o . and reconstituted in experiments using baculovirusrs recombinant ior

w r n HSV P gencs i 103). They included the DNA polymerase gene i LrL30i. UL42 which

increases polyrnrrase procrssivity. Ci29 which codes for ICP8 the single stranded bindinp

protrin. LyL9 the origin binding protein. and the helicaselprimase cornplex i C r L j . ULX and

1 5 5 2 t 95 i. t t wüs observed that long senomic concatrmrrs wrre pcnercited when the

rcplicaticiii cornplex consistrd of only the polymrriisc. ICP8 and the helicasdprimass

wbiinits. The facr that oris contnining plasmids in the presence of the LrL9 protein inhibitrd

rlie iorrriiition of thcse intermediates. suggests this rnechnnism occurs in an origin

inclcpcndent Prishion i t03 1.

I r i addition to the subunits dirrctly involveci in DNA replication. HSV codes tor ;i

rct of 13 enzymes irnplicated in dronynucleotide metabolism. Thymidine kinase iTKi.

rihonucleoiidc reductase (RR). uracil DNA glycosyiase. and deoxyuridinr triphosphate

nuclcotidohydroli~~e (dUTP&se) al1 function to ensure sufficient nuclcotide pools in non-

~ h i d i n g C C I I S for viral DNA synthrsis. Althou~h the.st. protein5 ;ire not functioniill)

rcq~iircd fur grou-th in rapidly dividing cells. thry appsar to be iniportitnt iu HSV

rc;icti\,rit ion from latenc y sincr terminal ly differentiatcd nrurons no longer maintain

dficicnt nucleotide resources required for virai DNA replication (95 i .

1.6 Regulation of Gene Expression in the Latent Cycle

Once the viral genome is uncoated and enters the nucleus of a neuronai ccll. a

conmitnient towards rither the lytic or latent cycle of infection occurs. Howrvrr. the

cellular iind/or viral fiictors that contribute to the activation of eithrr pathway remains

rinclciir. The detininp characteristic of HSV Iatency is a transcriptional shutdown of the

entirc viral penomr to a quiescent statr. such that the virus can establish a persistent

infcciion in the host (92. I 1 1 ). Wiih the exception of the LAT region. which grneratr viral

ir~inscripth throu~hout the course of latency. the genome rrmains nuclelited in a linrar or

circ~ilar configuration. This LAT region is locatsd in each of the invertcd repeats. and _oives

risr to the LATs (latency associated trinscripts) (95) (Fig. 1 .?A).

The LAT region contains two identified latency promoten. LAP! and LAP2. which

lire srparaird by approximately 1300 bp in the inverted repeat region of the grnome (95) .

The LXP2 promoter rrscmbles many eukaryotic housekreping grnc promoters and

produces (i large non-polyadenylated RNA of approxirnateiy 8.3 kb < I S . 11. 91. 108.

1 I 1 ) . This transcript is then altematively spliced into one major species of 2 kb and tu'o

niinor species of l.45 and 1.5 kb. The 1.0 kb RNA product appears to br a stable intron

genercitrd by the RNA processing rnachinery of the host. and remains intranuclear. Because

d its localization. the 2.0 kb transcript is thought to nrver be translatai into protein i 18.

2 1 . 92. 1 1 1).

The importance of the LAT region is perplexing to rrseürchcrs brcausc no linoun

t'iinc.ti~)ti hur cwr been uttributcd to this region of the virus genomc. Thc =tinerul conasnsuh

\r ah that thc LXTs wcre byproducts of nonspecific transcription initiated by the d l . The

! k t ihüt thc L M 2 promoter rssrmblrs rnany eukaryotic housekreping promoters also

rupportcd this view. In a number of genetic aniilyses of thc LAT rcgiun uhing inacrtiuiisl

~ i n d de lctiond niutiints. insignificant deficiencies were obscrvsd in the establishnirnt.

niciintcnance or reactivation properties of Iiitency (31. 54. 58. 110). Howrvrr. u

conrradictory body of evidence showed the potential existence of a translated protrin from

ihis region which significantly contributed to the reiictivation ability of HSV. In this study.

B H K selh ( baby hamster kidnsy stlibiy tr:insfeçted with ;i plasmid containmg the

II) porhetical LAT ORF showed a 2 foid increased frçquency of reactivation i 1 15 1.

Althoiigh this rvidencr dors not conclusively prove the existence of a translatrd protrin

t'rom the LAT region. some degree of importance to~vards virus reactivcition mciy be

attributrd to this region.

1.7 HSV-1 Vectors

HSV has many favourable characteristics that can be rxploited as a delivrry

mechanism for yrnetic rnaterial to cells. Because HSV is a large DNA virus that can

accommodate large amounts of foreign DNA within its gnome. a broad spectrum of vector

configurations have been developed which utilize the natural infrctivity of the viru to

drliver p r s to cells iti vivo and in vitro. At opposing ends of this spectrum svist the

helpsr-independent and helper-dependent HSV vector types. For the purpose of this thesis.

I have catrgorized al! HSV vectors based on their requirement for helper functions during

grun-th into one of thrse two types. Helper functions refer to al1 the rrms-acting factori

rç.yiiii+cd for rcplicÿtion and packaging of an HSV vector into irifeçtioi~i virianh. including.

\tructiirul protrins. enzymes for repiication and metabolism. as w r l l as. the rrgulatory

plutt'i113.

1.8 Helper-lndependent HSV Vectors

Of the entirc HSV genome. approuimately 45% of the cudiny scquenw 15

dirpcnsahlc (non-essential genes) and thus. not required for virus growrh in culturrd crllh.

hr ious c D M s can be recornbined into these regions. such that the virus ücts ii'; ü vector KI

delivcr and express forcign genes in crlls 17. -13. 64. 7 5 ) . These vectors. commonly

rcfcrred to as rccombinünt HSV vimses. are thus helper-independent becausr theu do not

requirc ;in) txterniil hrlpcr functions to grow in cultureci cells. Since the! contain ail the

g c ~ ~ c i i c information necessary to generiltr infectious parricles within ch<: vrctor bückbonr

iirclf. propagating a helper-independent vrctor in culture sirnpiy involves growing the virus

or1 a suitable cell line and harvesting the package vector panicles (Fig. 1 .-l1(7.13.64. 751.

The major advlintages associated with the use of recombinant HSV viruses are tuo

i;)ld: i 1 thc! can accommodate a significant amount of forr iy senstic material. and i i 1 ciin

bc propügatsd to high titre quite easily in culture ( > I O 9 vector particles per millilitre) (Table

1 - 1 1 0. 37. 7 5 ) . For example. it is possible to engineer a rHSV vector to sirnultaneously

eicpress f iw different cDNAs. including three soluble human cytokines (IL-:. GiCI-CSF.

;mi [FS--+ a T-cell CO-stimulatory molecule (CD80). and the HSV thymidine kinase gens

froni various locations in the viral genome (65). This flexibility allows for the utilizütion of

HSV helpcr-indrpendrnt vectors as delivery systems for cornplex thrriipeutic stratesies.

FiniilIl-. iinlikr integrating vectors, for example. systerns based on retrovirus or Irntivirus

FI(; 1.4. Hclpcr-indcpendçnt HSV vectors. Growth of the virus on a complrmenting ceil Iiiic amplifich the therapeutic genr which is integrated in the genome. Prozeny virions are t i~ i rwi tcd iind LIMA as lysrites to transduce türget cells. Since in most cases the virus is JcIc~cd f o r imc or more essential genes thesr vinisrs can express the grnr of inrertist but ciiiinor i-cplicatr in non-çornplernenting crll types.

Helper-lndependent HSV Vectors

Infection

Transducing Lysate

I 09~ fu / rn1 ( 1 0096 Vector)

-TABLE 1 . 1 Generril chriracteristics of HSV vectors

Hrlper - Helper - l3ependent Independent

(rHSV) (rHSV) ( ~ ~ o s m i d s c r i

Contains Viral Genes Y es ?JO No

Transgenr [ncorporation upto 15 kb upto l50kb upro l 5 0 k b

Rcpliciiticin-Defectic'e Yes Y es Yes

C! ioioxicit> Li Yes Helper Vin15 No

Rcyuirc\ Hclper Functionh Yo Y es Ys3

Hclper Virus Conrüminiition O. 1 -0.00000 1 92 25-90% 0.000 1 %

P ~ i c k a p i Vector Yieldc > 1 0') 1 O# 1 06

Cytotouicity as detrrmined by CPE criuscd in trrinsduçed cells due ro viral gene cxpressiun.

The rrquirement for HSV factors not supplicd by the crll linr for grow of the vrctor in culture

Y ield of vector infectious particles in transducing units per millilitre (t.u./rni)

hiolog. the threat of cellular transformation is lacking sincr the virus genoms is mainrainecl

ab ;in ;iutonomous episomr in transduced neurons (32 ).

The major problem associated with this vector format is the toxicity gcnrrated in

trsnsducrd cslls from leciky expression of HSV ornes lrom the vrctor backbone. This

phenurnenon has proven to be a significant obstacle to the utilization of recombinant HSV

L iruses as an expression platforrn for foreign genes becausr transduced çells quicklq

wccumb to cytopathic effects (CPE). One way to limit the toxicity induced by hslper-

independent vectors is to target neuronal ceIl types. In this system the toxicity of the vrctor

i h Ir.ssc.ned brcause the latent cycle shuts down almost al1 promoters in rhr. tector

Ixickhonc. i 109. 1 I 1 . 2 1 - 1 . 125 1. This phenotype is utilizrd in culturd crlls and

iiiiininl riiodcl.r to provide stable transgrne delivery and expression of nuwl protsins. I t htia

hcen hown in a number of studirs using nervr growth ixtor differentiaud PCI 2 cr l ls i rat

idrcnal phcochromcytoma). which serves as a mode1 system for the study of sympathetic

iicurons. thüt recombinant HSV vinises can be maintaincd in a non-producrive ini'eciious

\tate for u p to Y werks. In this statr. the quiescent genornrs produced no deisctable viral

mtigcns. and expression from the LAT region rrmained constant throughout. Howcver.

ihc persistence of thesr genomes often depends on the use of antiviral d rup likr xyclovir

o r acy cloguonosinc to obstruct virus replication. and thus inhibits productive viral

rcplication. This sugzrsrs that sent: expression from this platform iz po\biblc.. and support3

the i t t i l i ty of recombinant HSV vimses in neural experimrnts (31. 68.69).

Early attempts to achisve prolonged genc expression in the CYS of animal mode ls

wing a varicty of HSV-I strains have resultrd in only shon term detcction. Since thesr:

\i-usrs are reliltively virulent and maintain thcir replicative charricreristics. rnost rrltnsducrd

4 s do not survive because of leaky viral gene expression which leads to cellular toxicity

( 3 7 ) . This toxicity causes cellular death before the latency-associated shutdown of

transcription cün take affect. Therefore. forced virus entry into the latent cycle is probably

niandatory for stable genr expression and maintenance of recombinant HSV vinisrs.

One way to deal with this problem of cellular toxicity caused by unwantrd grne

expression is to delete various viral genes from the genome. panicularly the transactivatinp

u proteins because they induce the expression of the P and y senes ( 37 . 59. 75). Becausr

thesr wctors do not contain al1 the essential genrs. thçy have a replication-dekctive

phcnot>,pc and ncrd to be complemented for the missing factors in order to grou. In [hi\

iabc. the nwht common source of helper Functions are ccll linrs that stahly csprc55 the

riiihhirig genci 5 1. This allows for the vcçror to br propagüted ro high ritrr in culturc and

pro\ idch wfficicnt material for direct U r i i i w use (60-61. 75. 127). Ewn though thesr:

rcplic;ition-dcfecrivr vectors are drpendcnt on a smiill subser of viral frictors supplied b) the

packaging cell line. they still contain greater than 9 0 9 of the viral srnome wirhin the

w c ~ o r . and thiis retain most of the characteristics of recombinant HSV viruses. For this

rcwm 1 h w r , p lxrd the replication-defective vectors in the helper-independent ciitegory.

The first systrm of this kind preventcd the expression of P and y gencis by utilizing

an 1CP-l-delritrd replication-defective vector. In non-cornplementine crll types only ihr

renitiininl CL p e s ((127. uO. u71. and u47) are expressed. therefore. rhr toxicity , wnrlr;~[eii

h! th< 13 and genss is prevrntrd. Xeverthrless. the tramducrd crlls showrd signitïçiint

~osicity. such as alterations IO cell morphology and chromatin fragmentation (60. 6 1 . 70).

Han tiver. subsequrnt generations of replication-defective vectors that have mu1 tip le

ddetions in the u genes have made significant improvements in this area. The crown jervrl

of the group is a member id109) that contains deletions of svery a genes i u-l. u77. uO.

u22. and u-17) (971. In non-complementing ceIl lines. the dl09 genome typically cnters a

quiescent stiitr similar to latency. and green florescence protein (GFP) reporter sene

expression was drtrctsd in one third of the culture for at lrast 28 days in both Vrro and

human srnbryoniç lun? (HEL) cells (98). This pseudo-latent statr mistir5 the parametsr for

lou cytotoiricity because i t does not generate much CPE. and imparts prrsistrnce to the

term wctor DNA in non-neuronal ce11 types. However. attempts ro achieve high level Ion,

reporter genc expression from this platform were largely unsuccessful presumably dur to

ttic I;itcnq-associatecl shutdown of the vector genomr (53 . 55.56. 97. 98. i 26

.Attr.mpts to fxilitatr long rem gene expression in transduced crlls Lire onsoins and

iiiinisrous promoter configurations and regulatory elements are being investigatrd that

riippori suçcrssful expression profiles. One lrsson that has been takrn from the biology of

HSV is the iitil i ty of the LAT region of the genomc. Brcause the LAT rrgion of the v i w

gcnuiiic rerniiins transcriptionally active during the latent cycle. it may be possible to

cnginccr a vcctor thüt utilizes this region or its promoters (LAP I and L A P 3 as an

zuprcssion platform for prolonged gene expression. Significant progress hiis bren made in

ihis area. and by simply inserting an interna1 ribosomal entry site ( IRES, driving the

i.c.poiicr p c into the LAT resion. stable P-galactosi Jil\r rrpurtcr gcnr x t i i it! u ah Jetccicd

iip t o 307 düys postinfection. within neurons located in facial and hypoglossül nervr nuçlei

mi thc iipper cervical spinal cord (69). However. the number of cells that stained positive

itt ihc cnd of the study was significantly less çompared to the bqinning. and thereforc.

ilimption of the LAT region may not br compatible with genonie perbistrnct. and

rnüinrenancc long term gene expression (69).

Prcsently. the factors controlling the complete latency-associated shutdown of viral

gcne expression have not been clearly defined for HSV. and limiied progrrss has brcn

made in bypassing this shutdown to facilitate long trrm transduced _orne expression.

Thcretùrr. the utilization of recombinant HSV viruses as gene transfrr vehiçlss for long

icrm transzrnr expression has not been totally realized. Finally. the utility of helper-

independent vectors in senetic therapies represents ri potential risk to the patient by the

rsactivation of latent wiid c p e virus in vira.

In contrast to prolonged gene expression. recombinant HSV vinises have been

s h o w to br eftlcacious for tumour therapy. In this strategy the replicative lytic propertiçs

of the virus are utilized to cause cellular death of cancerous cells. or used to stimulate a

strniiç mi-tumour immune responss ( 1 1 . 13. 63. 1 16. 1 17. 120. 128 I. This approach is

brht illustrlited by recombinant vinisrs that are basrd on the HSV G107 strain. (3207

çarrics deletion mutations for both copies of the nruroviruirnce factor 734.3 gentt. and

ciddi tionül l y. have the bacterial locZ reporter sene insened into the ribonucleotidr rcductasc

gcne t LrL3YI of the virus. These mutations restrain the ability of the virus to grow on

non-replicating crll types such as neurons. because of a premature shut off of protrin

i!ntht.sis which prevents the translation of -{ gene rnRNAs ( 9 5 ) . Howcvsr. in rlipidl!

LI]! ;Jing ceil types. including turnour çells. the growth charücteristics o i G107 c«mplired ro

i t d d t i . p ~ HSV strains are indistinguishable. In thrse systcms. (3707 repliccite~ in turnour

c d 13 id c;i~iscs ceil death. whereas in non-tumour ( normal cclls. (3107 m e r s latrncy and

ihc wrgci C C I I S rcmilin viable. Thus. thrrapies büsed on G207 psrmissiveness have heen

in~.estiglited for neuronal tumours and other cancers. and pressntly human dinical trials

h a w been approvrd for the utilization of G207 as an oncolytic viral agent for human

intrxcrchrril tumours (57).

1.9 Helper-Dependent HSV Vectors

Hclpcr-dependent vrctors contain only the minimal viral replicon m i require

cwrniil hclpcr functions in order to srow. The minimal rrplicon Ib r HSV uah l i r h t

ideiitified in rxperiments that isolated the defective grnomes produced when HSV wos

wially passaged in cultured cells at high multiplicities of infection. Genetic tinalyjis of

these dttfectivr srnomes revealed two common rlements: an origin of replication ( oris). and

;i paçka~ing srquence (pcic) ( 106. 107). Whrn thrse two elrments cire cloned into bacteritil

plasmids and CO-transfected into cells dong with wild ype HSV. the plasmids c m be

rrpliccltcd and packaged into infectious vinons as head-to-tail repeat units of approxirnately

152 kb. Therefore. such plasmid vectors have been historically rrferred to as HSV

ampl icons.

HSV ümplicon vectors were first described by Spaete and Frenkrl in 1952 as a

cuknryotic defective-virus cloning vector. The First successhl gene transfer using HSV

amplicons socn followed with the expression of chimeric chicken ovalbumin gene in HEp2

crlls i 67 ). Since the viral replicon is so small (< 1 kb). and constitutes the only viral

rcqur:nct.> required in the vector. HSV amplicons have many advanrages owr rccimibinant

1 ~ruwi . Firsrly. srnrrating an HSV amplicon simply involves subcloniny the oris and ptcc

qLicncc into u conventioncil bacterial plasmid that contains a cDNA expression cassette.

C'rilikr: rccombinant viruses which requirc the cDNA construçt to br: recornbinsd into the

\,ii.;il gcnorne and subsequent virus isolation. ampliçon veçtor u n bt. -enrrica11!

iii;inipuliitcd much more rasily by standard recombinant DNA methodolo~ies i 3 2 ) .

Another advanrage of helper-dependrnt vectors is their ability to accommodate large

m u u n c of generic material. and in some cases ris rnuch as 1-10 kb. By virtur of the veçtor

baçkbone being only a small fraction of the size of helper-independent genomes. in rheory

iip tu 10 tinirs more forrign DNA can be inserted into an amplicon vector. FinaIl).

iin~plicon vectors contain no virally-encodrd genes which çan interfere with grne

c.;prc*sion of the trlinsgene. This riiminates the tonicity of unwantçd gene expression from

ihc vector ;ind provides a less toxic way to transducr cclls. Thrrefore. bcised on thrir vcctor

btiçkbonc configurations. amplicon vectors are cven more tlrxiblr and safer for gene

iranstèr protocois than recombinant HSV vimses (Table 1. I ) ( 15.39.53 .

The major problem associated with the use of HSV amplicons is the technicd

di fficulty of packaging these vectors into infectious virai particles. In order for an amplicon

wcror to be propagated as an infectious virus. a helper virus is required ro supply al1 the

necessiiry viral replication and packaging factors. The components of these methodologies

arc col lectiwly rrferred to as hrlper-dependrnt or amplicon packqing sytems. Early

\!stems utilized wild c p e HSV to provide helper function in infectrd cells that were

pruiously transfected with the amplicon vector (35. 107). The way this system works is

the packaging crlls are permissive to virus growth. and as a consequencr the vrctor is ülso

rcplicaied and packaged into infectious virions (Fig. 1.5Ai. Followin$ growth of the virus.

[ l ie partic1r.s produçed are harvrsted and used üs a transducing Iysate for target csll

infection. Howevrr. thrsr lysates contain two populations of virions. a fraction that contain

piickaged vector. usually 4 0 % . and the majority that contain helper virus grnomes. Since

purification of the packaged vector particles from the contaminating HSV virions is not

ph)sicall) possible. severe deiivery-üssociated toxicity and CPE is grnrraied in crlls

t«llo\ring vtxtor administration (7. 32. 52) . This toxicity stems from the contaminating

hclper virus in the lysatr and is not due to the packaged vrctor particles thcmselvrs. If a

replication comprtent helprr virus is used to package the amplicon. and the transducin~

I! >;LW IS utilizifd in an animal rnodel. the hrlper virus continues to rcpliclitr. and h p r d until

c i CS' trmiducrd csll is destroyed or the animals suffrr symptorns of disrase. The need to

liniit thc toiiicity of the helprr virus has driven the devrlopment of a variety of amplicon

p:icl;iiging y t r r n s .

The first practical packa@ng system urilized an ICP4-deletrd. replication-defective

HSV i d 120 or D30EBA) as a helper virus (74. JO). In this sysrem. rhe ümplicon vector is

transfeçted into an ICP4- cornplementing cell line. which is then super-infected with ci 110

or D30EBA. and the resulting infectious particles are harvestrd following srowth of the

virus. Although this approach can yield titres of packaged vrctor approachins 1 O 1 4

tr:iniduçing units per millilitre < t.u./ml). the transducing Iysates produced contriin ür k a t IO

fold niore d 120 virions. Even though the d 120 or D30EBA virions are replication-defcctiw

and ccinnot sprelid ro other çells. sisnificant CPE and irnmunolo=icai responses are

y-teratrd in the host by viral rintigens. such that cxprrirnsnts that test an- prolonged genr

cspression from an amplicon vector cannot be effectively rvaluated.

One sene therapy approach which kvas not restricted by the negativr rffrcts of

hslpsr virus contamination utilized an HSV amplicon vector for cytokine in vivo delivery in

FIG. I .5. Hclper-dcpcndent HSV vectors. A ) The unplicon is transfrçted into a cimiplciiicntirip crll line and which is then superinfected with a helper virus. The hrlprr \ ii-113 pro~~ides ail [he nrcessary factors iri tnrtis to replicate and package the amplicon vector into HSV- I infesiious particles. Following growth of the virus the infectious panicles are tiai.t~c~tcd iind used as lysate to transduce tiirget cells. Thrsr lysarcs are ri mixture of p;ickagcd tiniplicon and helper virus particles. B) Helper virus-frec transfection-basrd \ ~ i i c m . The series of five cosmid clones which contain the complete HSV-1 g n o m e i ~i~\ir i id .h sct is CO-transfected along with the amplicon vector into a suitable cell line. The cosniid 3c.t provides al1 the helper functions in tram required to generatr infectious HSV- 1 pu-ticlc\. Followinf growth of the virus the infectious panicles are harvested and used as Iysates io transducc target cells. Since the helper virus (cosrnidAn set) does not contain ciny ~ C K c lc'nlcnis on1 y the vector is packaged into infectious panicles.

A) Helper-Dependent HSV Vectors

( Virus-Based)

X r n ~ l ~ c o n 2% m s tr, Helper Virus \@-

Transfection, -_ Infection

. 1.- -

Canoiememed Vliai Genmes Helper Virus mg y s Gene Expression

-* - , DNA Cleavage Production o l ,

Transducing Lysate

I 09~ fu / rn1 ( C ? O O h Packaged Vector)

Helper-Dependent HSV Vectors (Transfection-~ased)

- \/

;. o q

Co-Transfection

' . rrris Jr'% ',

HSV cosmids recombine

Replication uac JI~S . J ~ L t .;

--/

--

Cancatemenxd Ampiicon Helper Virus , Gene Exgression ---

< >

/

Transducing Lysate

1 Oôpfu/rnl (Helper Virus-Free)

a cancer tumour animal mode1 (20). In this case the interirukin-2 i IL-2) sens was ussd to

htirnulütr ünti-tumour responses. speci ficall y. üntigen-speci fic cytoto~ic T lymphocyte and

non-sprcific lymphokine-activated killer cell responses. Male Fischer rats were usrd in the

w , d v that had been seeded subcutaneously with a tumour cell line derived lrorn a

mcthychollinthrene-induced squarnous cell carcinoma. Amplicon vectors werr packagcd

using D3OEBh as the helper virus and 4x106 HSV-IL7 panides were usrd p u injection.

Trutnicnt wi th HSV-IL2 vector resuitèd in a 7 told reduction in tumour volume for both

ilircctl! injc~.ir.d and non-injectrd tumoiin on the contralateral sidr 42 d q i into the qtudy.

Thih c t'kct correlateci with a significant increasc in sumival of the animds in the ILI ireüted

groiip compared to the controls (20). The presencç of contiiminating D30EBA virus in the

transduçing lysatr did not restrict the cfficacy of the study becausc the cxperimrntal goal

ii ;ir tu c;lu'ie cellular d a t h of tumour celis following gene transfer. S imiiar studies which

~ispendcnt on Li lling crlls are not severely affrcted by helper virus contaminarion since long

tcrm gcnc expression is not formaily required. Therefore. replicntion-drfective pückasing

\)stcnis have potrntial to provide sufficirnt packaged amplicon vector mütcrinl fur the

administration of such therapies in humans. Whrther or nor these Yrctors arc s l i k remliinh

tt:, bc ciciterniined.

Othcr repl ication-defective helper virus packagins sy stems have bern reportrd with

thc niost useful bring the one developed by Lim and colieagues t 7 7 ) . This paçknginp

y t c m consists of a replication-defective helper virus that is deleted in the ICP27 sene

i 5dl1.1). and a cell line. 7-2. that stably expresses ICP77. The lysates gencrated contain

comparable lrvels of packaged vector but significantly less helper virus. The ratio of helper

virus to packaged vector is typically about 1 . and thus becarnc the packaging sy stem of

choiçe for HSV amplicon vecton. However. even with thrsr irnprovements in vector ratio.

unlcis therr is a mcchanisrn in place to provide for the selective replication and packaging

of the amplicon vector over that of the helper virus. the toxicity and immunologica1

responsrs _ornrrated by the contaminating helper virus severely restncts the itr iivo efficacy

of amplicons (40. 77).

Such modifications have bern investigatrd and are refrrred to as "pigg),back"

packiiging prorocols. In thrsc systems the amplicon vector contains the essenrial genr that

ciwiplcrnents the replication-drfective helper virus. such that durinp the püçkaging reaction

oiil! cclli th;it have both an amplicon and a helper virus can seneratrd infectious particles.

This sirategy rfkctively prevrnts vinons being produced by çells not involveci in amplicon

pciçkiging. Howrver. even when seiective replication and packaging of the ampliçon vcctor

i h provided. the helper virus represents IO-25% of the total virus yield (89. 1301.

.-4dditionally. the replication pressure enened by the system selects for virusrs thlit hiive

rew-ted to a rrpl içation-competent phenotype by a single recombination event betwc.cn the

hdpcr virus gcnome and the amplicon. Evsntually. the lcvel of revertant v i r u m cscocd\

thc mioiini of packaged vector foilowing threc serial passages. and similar to rhr situation

u ith the ICP4 and ICP?7 replication-dcfrctive helper virus packaging systems. signifiant

Jclivsry-lissociritrd çytotonicity remains in the transducing Iysatrs. hlthough this problrm

ctin potrntinlly be eliminated through the use of rxtremely detoxified forms of HSV to

pi ickqr amplicons. for examplr. d 109 which contüins mutations in al1 five u senes. such

niutlints typicaliy grow much more slowly and the yield of packaged amplicon is

corrrspondingly lower (97.98). Finally. much like recombinant HSV viruses. the pressncr

of viral genomes in the transducing lysates (helper virus) represents a potrntial a risk to

patients ttirou_oh the reactivation of latent viruses or recombination with endogenou5 viruheh

c 15. 39. 5 2 ) .

A n slegant helper-dependent packaging system was described by Frarfel and

collrngues which yiclds transducing lysates free of helper virus contamination (3-11. This

beconci _osneration packaging system specifically addresses the cytotoxicity problem of the

prrceding mrthodologies (Table 1 . 1 ). Much libe the packaging protocols practiced for

retro~iral vectors (58). which consist of a series of plasmids that rncode the complète

hrlper virus grnome. a set of five overlapping cosmid clones of the wikl npr HSV grnome

iras niodiiied to provide the required helper funçtion ( 19). Cpon transfcction into a suitable

ceIl linr. grne rxprrssion from the fragrnented HSV helper virus (cosmids set) supplies al1

thc requircd factors in trtzns for vector replication and packaging (Fig. 1 . j B ) . By

rpecifically drleting the pczc elements in the tepetitive tr srquencrs of two of the cosmids.

ihc cosrnid set is rendered packaging-drfective and whçn CO-transfectsd into mlirnrnalian

i ~ l l \ dong with an HSV amplicon vector. only the latter is packagcd into maiiirr \ir;il

piirticlei i 34). The resulting lysares genèratrd from this approxh contüin as rnuch as I O h

i.~i./nil of paclsged amplicon with less than 10-6 plaque forming units of revenant hclper

tirus. Flot$-cwr. i t does not providc for any selective replication of the amplicon vector.

aiid this nia! be why the ovrrall y ield of packaged vector particles i?; qui te lou (Table 1 .1 ,.

Studies using helper virus-free lysates have sincr been reportrd. and hiive s h o w

u x n s f u l transduction and reporter gene expression in primary neural crlls in culture. and

thc central nrrvous system (CNS) of rats ( 1). Cultured primary rat striatiil cells wrrr stiibly

transduced by an HSV amplicon encoding the green tloresccnce protein iGFP> orne.

\\. i thout an'. apparent s i p of toxicity. and GFP expression remüincd rçlatively consunt

incr (i periud of S days. In cornparison. 1 x 105 vector particles werr injccted into the right

stri;iturn and right hippocampus of rats. and GFP reporter gene actîvity \vas assaycd 2 days

later to determine the transduction sfficiency of the vector kt i s i i - o . An cfficirncy of

approitimatrly 1.7 and 0.93% was seen in the hippoclimpus and striatum. respectivrly. as

detcrminrd b! the average nurnber of GFP positive cclls prr animal per vector particlç

inoçulatrd. Although no significant cytotoxic effects were observed. a higher dose of

packligrd vrctor must be administered in order to adequately test this result. Therefore.

grne transfer studies using HSV amplicon vectors packaged by the cosrnid method have

o n l ~ met limited success (17. 33. 41). The reason for this primarily items from the

inrfficicncy of this protocol to provide sufficient material For an in depth ünülysis of the

r ff içac y of the vector. Considering the aforementioned transduction e fficiency of HSV

aniplicons in the CNS and the packaging efficiency of the cosmid-based systrm. a more

iidvançcd trchnology is required to generate more material brfore (i succcsslul srnr therap'

mare-\ - - uring HSV amplicons c m be reiilized.

1.10 Objectives

The major limitation in the application of HSV arnplicons teçhnology io srne

thcrap) i the lack of a high efficiency packaging system whrrt. high titre .;toçks of

p~ickagcd vcctor frer of üny contaminating helper virus can br grncrüred. This inadeyutiq

in ohtaining cnough materilil to thoroughly test the efficacy of HSV amplicons in animal

niodcls has pressed researchers towards drveloping newer and more advünccd vector

piickiiging protoçols. The objective of this thesis was to construct such a systrm whrrrby

higher ~ i e l d s of packasrd vector can be obtainrd. The approach rakcn w u to rnodify rhe

cxi\iing cosrnids suçh that the inherent inefficicncy at providing profiçient helper funsrion

specifically addressed. A possible rerison for this is the fra_pmentrd gcnomr of the

co:-riiid\ does not provide a stable grne expression plütform for hrlper virus yrnr

r..cpression. and thçrefore lowers the efficiency of virion production. M y hypothesis is that

h' usiiig an intact helper virus genome as a closed circular molecule in the paçkaginp

rcciction. the y ield of packaged vector will surpass the nrnount generated by the fragmentrd

helpcr virus genorne of the cosmid s ystem in a transient transfection-based packaging

protocol. To test this hypothesis. I cloned the rntire packaging-defectiw HSV grnome of

the current system as a single infectious plasmid in bacteria as a bacterial artificiiil

chroniosome i BAC or bacmid ). Therefore. i t should be possible to increlise the arnount of

paçksgctd wctor using an HSV bacmid helper virus because this system provides a rnuch

more stable srne expression platform for the helper virus in the packasing ceil. My results

show that the rnhanced bacrnid system lessens the complexity and technical drmand of the

co\mid-bascd system and furthemiore. affords a Mold increasr in the yirtld of packagcd

\ t'c'ior.

REFERENCES

9 .

IO.

Ahoody-Guterman, K. S., P. A. Pechan, N. G . Rainov. $1. Sena- Esteves, . jacobs, E. Y. Snyder, P. Wild, E. Schraner, K. Tobler. 1. O . Breakefield, and C. Fraefel. 1997. Green fluorescent protein as a reporter for retrovirus and helper vims- free HSV- 1 arnplicon vector-mediated grne transfer into neural cells in culture and in vivo. Ncuroreport. 8:3801-8.

Alconada, A., U. Bauer, B. Sodeik, and B. Hoflack. 1999. Intracellulx traffiç of herpes simplex virus glycoprotein CE: charactrrization of the sortin_o signals required for its trans-Golgi network localization. J Virol. 73577-87 .

Bataille. D., and .A. L. Epstein. 1997. Equimolor seneration ut- the four pohsiblt: arrangements of adjacent L components in herpes sirnplen virus rype 1 rcplicotive intenediates. J Virol. 71:7736-43.

Bataille. D.. and A . L. Epstein. 1995. Herpes 4rnple.; tmirus type 1 rcplication and rrcombination. Biochimie. 773787-95.

Boehmer. P. E., and 1. R. Lehman. 1997. Herpes siniplex t*irus D ' i h replication. Annu Rev Biochcm. 66:347-54.

Borchers, K., M. Goltz, and H. Ludwig. 1994. Gcnomt: orgiinization of the herpcsviruses: minireview. Acta Vet Hung. 4 2 2 17-75.

Breakefield. X. O., and N. A. DeLuca. 199 1 . Hcrpes simplex virus for cene delivry to neurons. New Biol. 3:2O3- 18. C

Bruni. R., B. Fineschi, W. O. Ogle, and B. Roizman. 1999. .A novrl cellular protein. p60. interacting wiih both herpes simplex viru:. I regulator! psutetns ICPX and ICPO is modified in a dl-type- specific münncr and 13 rcicniited to the nucleus ~tfter infection. J Virol. 73:38 t0-7.

Cüi. W.. and P. A. Schaffer. 1992. Herprs simplrx virus type 1 ICPO rcgulntcs expression of immediate- rarly. early. and latr senes in productive1 y inkcted ceils. J Virol. 66:29O-C- 15.

Campadelli-Fiume, G., D. Stirpe, A. Boscaro, E. Avitabile. L. Foa- Tomasi, D. Barker, and B. Roizman. 1990. Glycoprotcin C-drprndenr attachment of herpes sirnplex vims to susceptible cells leading to productire infection. Virology. 1782 13-22.

Carew, J. F., D. A. Kooby, M. W. Halterman, H. J. Federoff. and Y. Fong. 1999. Selcctive infection and cytolysis of human head and nrck squarnous cell carcinoma with spüring of normal mucosa by a cytotoxic herprs simplex virus type I (G207). Hum Gene Ther. 10: 1599-606.

Carter, K. L., and B. Roizman. 1996. The promorer and transcriptionai unit of a novel herpes sirnplex virus I alpha gene are contained in. and cncodr a protcin in framr with. the open reüding frame of the alpha 72 gene. J Virol. 70: 172-5.

Chahlavi. A., S. Rabkin, T. Todo, P. Sundaresan, and R. Martuza. 1999. Effect of prior exposure to herpes simplex virus 1 on viral vector- mediated turnor therapy in irnmunocompetent mice. Gene Ther. 6: 175 1 - 1758.

Chen, J.. and S. Silverstein. 1991. Hrrpes simplex virusrs wirh mutations in the senc cncodins ICPO are defective in gene expression. J Virol. 66:IC) 16-27.

Chiocca. E. A., B. B . Choi, W. Z. Cai, N. .A. DeLuca. P. -4. Schaffer, M. DiFiglia, X. O. Breakefield, and R. L. 'ulartuza. 1990. Trlinder and expression of the lac2 gene in rat brain neurons rnediatrd by hçrprs riniplex virus rnutünrs. New Biol. 2:739-16.

Chou, J., and B. Roizman. 1985. Isomerization of hcrpes simplex virus 1 csnoine: identitïcütion of the cis-acting and recombination iitcs within the doniain ;f the î srquence. Crll. J1:803- 1 1 .

Costantini, L. C., D. R. Jacoby, S. Wang, C. Fraefel, X. O. Breakefield, and 0. Isacson. 1999. Gene transfer to the nigrostrinial sy stem bv hvbrid herpes sirnplex virus/adeno-associated virus amplicon vrctors. Hum & n i Ther. 10:148 1-94.

Croen. K. D., J. M. Ostrove, L. J. Dragovic. J. E. Smialeli. and S. E. Straus. 1987. Latent herpes simplex virus in human trigeminal sanglia. Dctection of an immediate early genc "anti-sense" trünscript by in situ hybridizütion. N Engl J Mcd. 317: 1427-32.

Cunningham, C., and A. J. Davison. 1993. A cosmid-bascd system for constnicting mutants of herpes simplex virus type 1. Virol. 197: 1 16- 124.

D'.Angelicü, M., H. Karpoff, M. Halterman, J. Ellis. D. Klimstra. D. Edelstein, M. Brownlee, H. Federoff, and Y. Fong. 1999. In vivo intsrlrukin-2 grne therapy of established tumors w ith herpes sirnplex amplicon vec tors. Cancer Immun01 Immunother. 47265-7 1.

Deatly, A. M., J. G . Spivack, E. Lavi, D. R. d. O'Boyle, and S. W. Fraser. 1988. Latent herpes simplex virus type I transcripts in peripheral and central nervous system tissues of mice map to similar regions of the viral genomr. J Virol. 62:719-56.

Deiss, L. P., J. Chou, and N. Frenkel. 1986. Functional domains within the a sequence involved in the cleavage- packaging of herpes simplrx virus DNA. J Virol. 59:605- 18.

Deiss. L. P.. and X. Frenkel. 1986. Herpes simplrx virus amplicon: clravage of concatemerïc DNA is linked to packaging and involvrs amplification of the trrminüliy reiterated a sequencr. J Virol. 57:933-4 1.

DeLuca. N. A., -4. M. McCarthy, and P. A. Schaffer. 1985. Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the genr rncoding immediate-eari y regulatory protein ICP4. J Virol. 56:558-70.

Desai, P.. F. L. Homa, S. Person, and J. C. Glorioso. 1994. A genetic sslrction method for the transfer of HSV-1 glycoprotein B mutations from plasmid to the viral genornr: preliminary characterization of transdorninance and entry kinerich of mutant virusrs. Viroiogy. 204:3 11-32.

Elias, P., C. M. Gustafsson, and 0. Hammarsten. 1990. The origin binding protein of herpes sirnplex virus 1 binds cooperatively to the viral origin of replication oris. J Bi01 Chem. 265: 17 167-73.

Everett, R. D. 1984. A detailed analysis of an HSV- 1 early prornoter: sequrncrs involvrd in trans-activation by viral immrdiatr-eürly genr products are not rürly- ucnr sprçific. Nucleic Acids Rrs. 123037-56. L

Everett, R. D. 1984. Trans activation of transcription by herpes virus products: requiremrnt for two HSV-1 immediate-riirly polypeptides for maximum activity. Embo J. 3:3 135-4 1 .

Everly, D. Y.. .Ir.. and G. S. Read. 1997. blutaiional an;ily\is of thc iirion huit bhutoff gsnc ( CL4 1 ) of hrrpes simplex virus ( HSV ): chüracreriziirion of HSV t y c i i HSV- I VHSV-2 chimeras. J Virol. 71:7 157-66.

Faber. S. W., and K. W. Wilcox. 1986. Association of the hrrprs simplrx virus r e & m r y protein ICP4 with specitic nucleotidr sequrnces in DNA. Nuciric Acids Res. 14:6067-83.

Fareed. $1. U., and J. G. Spivack. 1994. Two open reüding t'rümes @RF1 and ORF2) within the 2.0-kilobase latency- assocititrd transcript of herpes simplttx virus type 1 are not essential for reactivation from latency . J Virol. 685307 1-8 1 .

Fink, D. Je, N. A. DeLuca, W. F. Goins, and J. C. Glorioso. 1996. Gene transfer to murons using herpes simplex virus-based vectors. Annu Rrv Neurosci. 19265-87.

Frnefel, C.. D. R. Jacoby, C. Lage, H. Hilderbrand. J. Y. Chou. F. W. N t , X. O . Breakefield, and J. A. Majzoub. 1997. Gent. trmsfer into hcpatoçytrs rnediated by hrlper virus-frer HSVIAAV hybrid vrctors. Mol bled. 3:s 13-25.

Fraefel. C., S. Song, F. Lim, P. Lang, L. Yu, Y. Wang, P. Wild, and .A. 1. Geller. 1996. Helper virus-free transfer of hrrprs simplrx virus type 1 plasmid vrsctors into neural cells. J Virol. 70:7 190-7.

Frenkel. N., O. Singer, and A. D. Kwong. 1994. Minireview: the hrrpcts simplrx virus amplicon--a versatile defective virus vector. Gens Thsr. 1 Suppl I:S40-6.

Fruh. K., K. Ahn, H. Djaballah, P. Sempe, P. M. van Endert, R. Tampe, P. A. Peterson, and Y. Yang. 1995. A viral inhibitor of peptide transporters for antigen presentation. Nature. 3754 1 5-5.

Gaffney, D. F., J. McLauchlan, J. L. Whitton, and J. B. Clements.

1985. A modular system for the assay of transcription rrgulatory signals: the srqiicnce TAATGARAT is required for herpes simplex virus immediüte early senr activation. Nucleic Acids Res. 13:7847-63.

Galocha, B., A. Hill, B. C. Barnett, A. Dolan, A. Raimondi, R. F. Cook, J. Brunner, D. J. McGeoch, and H. L. Ploegh. 1997. The active site of ICP47. a herpes simplex virus-encoded inhibitor of the major histocompatibility cornplex (MHC)-encoded peptide transporter associatsd with üntigcn processing (TAP). maps to the NH7-terminal 35 residurs. J Exp bled. 185: 1565-72.

Celler. A. 1. 1988. A nrw mrthod to propagcite drfectivr: HSV- I vrctors. Sudeic Acids Res. 165690.

Çeller. .A. 1.. K. Keyornarsi. J. Bryan. and .A. B. Pardee. 1990. An ct'ficient dslrtion mutant packaging system for defrçtive herpes simpleu virus wctors: potential applications to human gcne therapy and neuronal physiology. Proc Nat/ Acad Sci L? S A. 8753950-4.

Geller, .A. 1.. L. Yu, Y. Wang, and C. Fraefel. 1997. Hrlper virus-frer hcrpes simplex virus- I plüsrnid vectors for gene thcrapy of Parkinson's discase and ot her neurologicd disorders. Exp Neurol. 144:98- 1 O?.

Gelman. 1. H., and S. Silverstein. 1985. Identification of immediate sarly senes from herpes simplex virus that transactivate the virus thymidine kinase sene. Proc Nat1 Acad Sci U S A. 825265-9.

Glorioso, J. C., W. F. Goins, D. ,J. Fink, and N. A. DeLuca. 1994. Hcrpes simplc.t virus vcctors and genc transfer to brüin. Dcv Bioi Stand. 8 2 7 9 - 5 7 .

Goldsmith, K.. W. Chen, D. C. Johnson, and R. L. Hendricks. 19%. Infectcd cell protein (ICP)47 rnhances hrrpçs simplex virus neurovirulencr by bloçking the CD8+ T ceIl response. J Exp iMrd. 187% 1-8.

Gustafsson, C. kt.. M. Falkenberg, S. Simonsson. H. Valadi. and P. Elias. 1995. The DNA ligands influence the interactions brtwern the herpcs impl r s virus i origin binding protein and the single strand DNA-binding protrin. ICP-8. J Biol Chem. 270: 19038-34.

Hammarsten, O., P. Elias, and N. D. Stow. 1996. Characterizrition of li binding site for the herpes simplex virus type 1 UL9 origin-binding protein within the CL9 gene. J Gen Virol. 77:969-76.

Hardy, W. R., and R. M. Sandri-Goldin. 1994. Herpes sirnplrx virus inhibits host ccll splicing. and regdatory protein iCP27 is required for this effsct. J Virol. 68:7790-9.

Hernandez, T. R., R. E. Dutch, 1. R. Lehman. C. Gustafsson, and P. Elias. 1991. mutations in a herpes simplex virus type 1 origin that inhibit interaction with orizin-binding protein also inhibit DNA replication. J Virol.

Herold. B. C.. and P. G. Spear. 1994. Nromycin inhibit3 glqcoprutrin C igC\-Jeprndrnt binding of hcrpes simplrx virus type 1 to ç r l h aiid a lw inhibit3 postbinding rvcnts in entry. Virology. 203: 166-7 1.

Herold, B. C., R. J. Visalli, N. Susmarski, C. R. Brandt, and P. G. Spear. 1994. Glycoprotein C-independent binding of hrrpes simplex virus to cells rrquirrs crll surface heparan sulphate and glycoprotein B. J Grn Virol. 75: 12 1 1 - 7 7 --.

Hill. A.. P. Jugovic, 1. York, G. Russ, J. Bennink. .J. Yewdell. H. Ploegh, and D. Johnson. 1995. Herpcs simplex virus turns off the ThP to svadr host immunity. Nature. 3754 1 1-5.

Ho, D. 1993. Amplicon-based herpes simplex virus vectors. iveth. Crll Biol. 43: 191-210.

Ho. D. Y., S. L. Fink, M. S. Lawrence. T. J. Meier. T. C. Saydam. R. Dash, and R. M. Sapolsky. 1995. Herpes simplrx virus vector ipstrrn: :in;ilysis of its in vivo and in vitro cytopathiç rfkcts. J Nrurosci blrthucl. 57:205- 1s.

Ha. D. Y., and E. S. Mocarski. 1989. Herpes simpleu virus latent RNA ( L A T ) is not rrquired for latent infection in the rnouse. Proc Nat1 Acad Sci C; S A. 86:7596-600.

Ho. D. Y., T. C. Saydam, S. L. Fink, hl. S. Lawrence. and R. 'il. Sapolsky. 1995. Defeciive herpes simplen virus vectors expressing the rat brain ducose transporter protect cultured neurons from necrotic insults. J Neurochem. k3-12-50.

Huard, J.. W. F. Goins, and J. C. Glorioso. 1995. Herprs simplex virus type 1 vector mediatrd gene transfer to muscle. Gene Ther. 2385-92.

Hunter, W. D., R. L. Martuza, F. Feigenbaum, T. Todo, T. Mineta, T. Yazaki, M. Toda, J. T. Newsome, R. C. Platenberg, H. J. Manz. and S. D. Rab kin. 1999. Attenuated. replication-competrnt hcrpcs sirnplex virus type 1 mutant G307: safety rvaluation of intracerebral injection in nonhurniin primates. J Virol. 73:63 19-16.

Javier. R. T., J. G. Stevens, V. B. Dissette. and E. K. Wagner. 1988. A herpes simplex virus transcript abundant in latrntly infectcd neurons is dispensable for establishment of the latent state. Virology. 166254-7.

Johnson, P. A., and T. Friedmann. 1994. Replication-defectivr recombinant herpes simplex virus vectors. Methods Ce11 Biol. 43 Pt A:21 1-30.

Johnson, P. A., A. Miyanohara, F. Levine, T. Cahill, and T. Friedmann. 1992. Cytotoxicity of a replication-defective mutant of herpes sirnplcx virus type 1. J Virol. 662952-65.

Johnson. P. A., M. J. Wang, and T. Friedmann. 1994. Improvèd cell wrvival by the reduction of immediate-early gens expression in replication- defective mutants of herpes simplex virus type 1 but not by mutation of the virion host shutoff function. J Virol. 68:6347-62.

Johnson, P. A., K. Yoshida, F. H. Gage, and T. Friedmann. 1992. ETfects of gene transfer into cultured CNS neurons with a replication- defectivç herpcs simplex virus type 1 vector. Brain Res Mol Brain Res. 12:95-102.

Kooby, D. A., J. F. Carew, M. W. Halterman, J. E. Mack, J. R. Bertino, L. H. Blumgart, H. J. Federoff, and Y. Fong. 1999. Oncolytic v i r d thcrapy for human colorectal cancer and l i ver metastases using a multi-mutatcd herpes simplex virus type- 1 (G107). Faseb J. 13: 13253.1.

Krisky. D. M., P. C. ~Iarconi, T. Oligino, R. J. Rouse. D. J. Fink. and J. C. Glorioso. 1997. Rapid method for construction of recombinant HSV sene transkr vectors. Gene Ther. 4: 1 120-5.

Krisky. D. 1 . P. C. Marconi, T. J. Oligino, R. J . Rouse, D. J. Fink, J . B . Cohen, S. C. Watkins, and J. C. Glorioso. 1998. Developmrnt of herpes sirnplex virus replication-dcfective rnultigenr: vcctors for combination gene therapy applications. Gene Thrr. 5: 15 17-31.

Kristie. T. M., J. L. Pomerantz, T. C. Twomey, S. A. Parent, and P. A . Sharp. 1995. The cellular CI factor of the herpes sirnplex virus enhancrr coinplrx is a family of polypeptides. J Biol Chrm. 270:4387-94.

Kwong, A. D., and N. Frenkel. 1985. The herpes simplex virus amplicon. IV. Efficient expression of a chimeric chicken ovalburnin gene ampli fied wirhin Jçfectivc virus genomes. Virology. 142:411-5.

Lachmann. R. H., and S. Efstathiou. 1997. The use of herpes sirnplcix virus-bnsrd vectors for gene delivery to the nervous systern. Mol 41rd T o J ~ ) . 3:JO-l- 1 1.

69. Lachrnann, R. H., and S. Efstathiou. 1997. Utilization of the herpcs siniplex virus type 1 Iritency-associated regulatory region to drive stiibls reporter zenr expression in the nrrvous system. J Virol. 71:3 197-207. C

70. Lachmann, R. H., M. Sadarangani, H. R. Atkinson, and S. Efstathiou. 1999. An analysis of herpes simplex virus gene expression durin2 Iiitency establishment and reactivation. J Gen Virol. 80: 127 1-82.

7 1 . Lai. J. S., and W. Herr. 1997. Interdigitated residues aithin a small resion of VP 16 interact with Oct- 1 . HCF. and DNA. iMol Ceil BioI. 17:393746.

7 1 . Laquerre, S., R. Argnani, D. B. Anderson, S. Zucchini. R. Manservigi, and J. C. Glorioso. 1998. Heparan sulfate proteoglycan binding by hçrpes simplex virus type 1 glycoproteins B and C. which differ in thcir contributions to virus attachment. penetration. and cell-to-ceIl spread. J Virol.

SI.

Lee. S. S.. and 1. R. Lehman. 1999. The interaction of herpes simple:< type 1 virus origin-binding protein (UL9 protein) with Box 1. the high iiffinity slerncnt of the viral origin of DNA replication. J Bi01 Chem. 274: 186 13-7.

Lee, S. S.. and 1. R. Lehman. 1997. Linwinding of the box 1 dement of a herprs simplex virus type 1 origin by a complrx of the viral origin binding protein. \iriglc-strand DNA binding protein. and single-strmded DNA. Proç Yatl Acad Sci C S A. 94:2538-42.

Leib, D. A.. and P. D. Olivo. 1993. Genr drlivery to ncurons: i herpss simp1e.c virus the right tool for the job'? Bioessays. 155-17-54.

Ligas. M. W., and D. C. Johnson. 1988. A herpes simplex virus mutant in which _olycoprotein D sequences are replaced by betagalactosidase sequences binds io but is unable to penetrate into cells. J Virol. 62: 1486-94.

Lim. F., D. Hartley, P. Starr, P. Lang, S. Song, L. Yu, Y. Wang. and A. 1. Geller. 1996. Ceneration of high-titer defective HSV- I vectors using an IE 2 dçlçiion mutant and quantitative study of expression in cultured cortical cells. Biotechniques. 20:460-9.

Liptak. L. M., S. L. Uprichard, and D. $1. Knipe. 1996. Functiond ordcr of asscmbiy of hrrpes simplex virus DNA replication proteins into prerepliçative site structures. J Virol. 70: 1 759-67.

Loçkshon, D.. and D. .A. Galloway. 1986. Clonin'r and charrictcrizlition of C

oriL2. a Inrge palindrornic DNA replication origin of herpes simples virus type 1. J Virol. 5 8 5 13-2 1.

Lycke, E., M. Johansson, B. Svennerholm, and L'. Lindahl. 1991. Binding of herpes simplex virus to cellular heparan sulphate. an initial strp in the adsorption process. J Grn Virol. 72: 1 1 3 1-7.

Makhov, A. M., P. E. Boehmer, 1. R. Lehman, and J. D. Griffith. 1996. The herpes simplex virus type I origin-binding protetn carries out origin specific DNA unwinding and forms stem-loop structures. Embo J. 15: 1712-50.

Slartin. D. W., and P. C. Weber. 1996. The a sequencr is dispensable for isornerizütion of the herpes simplex virus type 1 genome. 1 Virol. 70:8801- 12.

Michael. Y., D. Spector, P. Mavromara-Nazos, T. SI. Kristie. and B. Roizman. 1988. The DNA-binding propenies of the major regulatory protein alpha 4 of herprs simplex viruses. Science. 239: 153 1-4.

Msra. V., S. Walter, P. Yang, S. Hayes, and P. O'Hare. 1996. Conformational alteration of Oct-1 upon DNA binding dictates selectivity in differential interactions with related transcriptional coactivators. Mol Crll Biol. l6:WM- 13.

Montgomery, R. I., M. S. Warner, B. J . Lum, and P. G. Spear. 1996. Herpes simplex virus- 1 entry into cells mediated by a novel rnember of the TNFmGF receptor farnily. Cell. 87:4?7-36.

Papavassiliou, A. G., K. W. Wilcox, and S. J. Silverstein. 1991. The interaction of ICP4 with celllinfected-ce11 factors and its srate of phosphorylntion modulate differential recognition of leader sequences in herpes sirnplea vims DNA. Embo J. 10:397-406.

Paterson, T., and R. D. Everett. 1990. A prominent serine-riçh rrgion in Vmw 175. the major transcriptional regulator protein of herpes sirnplex virus type 1. is not essential for virus growth in tissue culture. I Gen Virol. 71: 1775-53.

Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993. Production of high-titer helper-free retrovinises by transirnt trrinsfection. Proc Niitl Acrict Sci L S S. 90:8392-6.

I'echsn. P. 4.. XI. Fotaki, R. L. Thompson, R. Dunn. SI. Chase. E. A. Chioçca, and X. O . Breakefield. 1996. A novcl 'piggybaçk' pacbaging y r e m for herpes simplex virus amplicon vectors. Hum Grne Thrr. 7:1003- 13.

Preston, C. M., and M. J. Nicholl. 1997. Repression of sene expression upon infection of cells with herpes sirnplcn virus type I mutants impairrd for immedilite-early protein synthesis. J Virol. 71:7507- 13.

Rice, S. A., L. S. Su, and D. M. Knipe. 1989. Herpes sirnplcx virus alphri protcin ICP37 possesses separable positive and negative regulütory activitics. J Virol. 63:3399-407.

Rock, D. L., A. B. Nesburn, H. Ghiasi, J. Ong, TD L. Lewis, J. R. Lokensgard, and S. L. Wechsler. 1987. Detection of latency-relatrd viral RNXs in trigeminal ganglia of rabbits latently infected with herpes simplrx virus type 1 . J Virol. 61:3870-6.

Roizrnan, B. 1979. The structure and isomerization of herpes sirnplex virus senomes. Csll. 16:48 1-94.

Roizman, B., T. Kristie, J . L. McKnight, Y. Michael, P. SIûvromara-Nazos, and D. Spector. 1988. The triins-activation of herpes irnplrx virus gene expression: cornparison of two factors and their cis sites. Biochimie. 70: 103 1-43.

Roizman, B., A. B. Sears, and R. J D Whitley. 1996. Fields Virology. Third ed. Lippincott-Raven. New York.

Sacks, W. R., and P. A. Schaffer. 1987. Deletion mutants in the gene rncoding the herpes simplex virus type 1 immediate-early protein [CPO rxhibit inipaired growth in ce11 culture. J Virol. 612329-39.

Samaniego, L. A., L. Neiderhiser, and N. A . DeLuca. 1998. Persistence and expression of the herpes simplex virus genorne in the absence of

immrdiate-early proteins. J Viroi. 72:3307-10.

106.

I 07.

1 os.

1 IO.

Samaniego, L. A., N. Wu, and N. A. DeLuca. 1997. The herpes simplen virus immrdiate-early protein ICPO affects transcription from the viral genome and infected-ceIl survival in the absence of ICP4 and ICP27. J Virol. 71:46 14-25.

Sarrias, $1. R., J. C. Whitbeck, 1. Rooney. L. Spruce, B. K. Kay. R. 1. Montgomery, P. G. Spear, C. F. Wnre. R. J. Eisenberg. G. H. Cohen, and J. D. Lambris. 1999. Inhibition of herpes simplsx virus ZD and I ymphotoxin-alpha binding to HvsA by peptide antagonists. J Virol. 73568 1-7.

Severini. A., A. R. Morgan, D. R. Tovell, and D. L. Tyrrell. 1994. Study of the structure of replicative intermediates of HSV-1 DNA by pulscd-field gel electrophoresis. Virology. 200:428-35.

Severini, A., D. G. Scraba, and D. L. Tyrrell. 1996. Brançhed $tructurcs in the intracellular DNA of herpes simplrx virus type 1. J Virol. 703 169-75.

Shieh. 1 T., D. WuDunn, R. 1. Montgomery, J. D. Esko, and P. G . Spear. 1992. Cell surface receptors for hrrpes simplex virus are hepliran sulfate proteoglycans. .J Cell Biol. 116: 1273-8 1 .

Skaliter. R.. and 1. R. Lehman. 1994. Rolling circle DNA replication in vitro bp a complcx of herpes simplex virus type 1 -rncodrd enzymes. Proc Nat1 Acad Sci L' S A. 91:10665-9.

Smiley. J . Rb, C. Lavery, and Al. Howes. 1992. The h e p 5 simplra virub tppt: 1 (HSV- 1 a sequence serves as a cleavage/packao_ing signal but does no[ drive recoinbinational p o m c isomrrization whcn i t is inserted into the HSV-2 senome. J Virol. 66:7505- 10.

Sodeik, B., M. W. Ebersold, and A. Helenius. 1997. Microtubule- niediatrd transport of incoming herpes simplex virus 1 capsids to the nucleus. J Csll Biol. 136: 1007-21.

Spaete, R. R., and N. Frenkel. 1982. The herpes simpleu virus amplicon: a n w cucliryotic defective-virus cloning-iirnplifying vector. Crll. 30:795-30-1.

Spaete, R. R., and N. Frenkel. 1985. The herpes simplex virus ümplicon: analyses of cis-acting replication functions. Proc Nat1 Acad Sci Li S A. 82:694-8.

Spivack, J. G., and N. W. Fraser. 1987. Detection of herpes simplex virus type 1 transcripts during latent infection in mice [published erratum appears in J Virol 1988 Feb:62(2):663]. J Virol. 61:384 1-7.

Starr. P. A., F. Lim, F. D. Grant, L. Trask. P. Lang, L. Yu. and -4. 1. Geller. 1996. Long-tem persistence of defective HSV- 1 vecton in the rat brain is dsmonstrated by reactivation of vector gene expression. Grnr Ther. 3:6 15-3 .

Steiner, 1.. J. G. Spivack, R. P. Lirette. S. M. Brown, A. R. MacLean. J. H. Subak-Sharpe, and N. W. Fraser. 1989. Hcrpes simplex

1 I I .

112.

virus type 1 latency-associated transcripts are evidentl y not essential for latent infection. Embo J. 8505- 1 1.

Stevens. J. G. 1989. Human hrrpesviruses: a consideration of the Iütrnr statr. ~licrobiol Rev. 53:3 18-32.

Stow, Y. D. 1982. Localization of an origin of DNA replication within the TRSIIRS repeated region of the herpes simpÏex virus type 1 genornr. Embo J . 1 :563-7.

Stow, N. D., and E. C. Stow. 1986. Isolation and characterizarion of ü

hcrpcs simplex vims type I mutant containing a delction within the gene cincoding the immediate early polypeptide Vmw 1 10. J Gen Virol. 67257 1-85.

Terry-Allison, T., Rv 1. !Montgomery, J. C. Whitbeck, R. Xu. G. H. Cohen, R. J. Eisenberg, and P. G. Spear. 1998. HvcA (herpesvirus entry niediaior A). a coreceptor for herpes simplex virus entry. also participates in virus- induced ceIl fusion. J Virol. 72:5802-10.

Thomas, S. K., Gv Gough, D. S. Latchman, and R. S. Coffin. 1999. Herpes simplsx virus lateiicy-associated transcript encodes a protrin whiçh preritl) enhances vims growth. can compensûte for deficiencies in immrdiate-early $ene cnprcssion. and is likely to function during reactivation from virus Iatency. J Virol. 73:66 18-25.

Todü. XI.. S. D. Rabkin, H. Kojima. and R. L. hlartuza. 1999. Hrrpes simplcn virus as an in situ cancer vaccine for the induction of specific anti-tumor imniunity. Hum Gene Ther. 10:385-93.

Toda, M., S. D. Rabkin, and R. L. Martuza. 1998. Tresitrnent of human breast cancer in a brain metastatic mode1 by G207. a repiication-competent multimutated herpes simplex virus 1. Hum Grne Ther. 9:2 177-85.

Vaughan, P. J., K. J. Thibault, M. A. Hardwicke. and R. .CI. Sandri-Goldin. 1991. The herpes simplex virus immrdiats elirly protrin ICP27 encodes a potential metal binding domain and binds zinc in vitro. Virology. 189:377-84.

Vlazny. D. A., and Nv Frenkel. 198 1 . Replication of hcrpes simplex virus DNA: localization of replication recoyition signais within defective virus genomrs. Proc Nat1 Acad Sci U S A. 78:742-6.

Walker, J. R.. K. G . McGeagh, P. Sundaresan, T. J. Jorgensen. S. D. Rabkin, and R. L. Martuza. 1999. Local and systemic therapy of human prostate adenocarcinorna with the conditionülly replicating herpss simplrx virus vector G207. Hum Gene Ther. 10:2237-43.

Wilcox, C. L., L. S. Crnic, and L. 1. Pizer. 1992. Replication. latent infection. and reactivation in neuronal culture with a herpes simplex vins thymidine kinase-negative mutant. Virology. 187:348-52.

Ct'ilcox, C. L., and E. $1. Johnson. Jr. 1988. Characterimion of ntirve crowth factor-dependent hrrpes sirnplex virus latency in nsurons in vitro. J Virol. iik393-9.

Wilcox. C. L., and E. M. Johnson, Jr. 1987. Nervr srowth factor deprivation results in the reactivation of latent herpes simplex virus in vitro. J Virol. 61:231 1-5.

Wilcox, C. L., R. L. Smith, R. D. Everett, and D. Slysofski. 1997. The herpes simplex virus type 1 immediate-early protein ICPO is necessary for the efficient establishment of latent infection. J Virol. 71:6777-55.

WiIcox, C. L., R. L. Smith. C. R. Freed, and E. M. Johnson, Jr. 1990. Ncrve growth factor-dependence of hrrpes simplcx virus Iiitency in periphcrlil sympathetic and srnsory nrurons in vitro. J 'leurosci. 10: 1768-75.

Wu, N., S. C. Watkins, P. A . Schaffer, and N. A. DeLuca. 1996. Prolongrd gcnr expression and crll survivül lifter infection by a herpes .;implci.x virus mutant drfective in the immediate-early grnrs rncoding ICP-I. ICP27. and ICP22. J Virol. 70:6358-69.

Wu. X., Y. Leduc, M. Cynader, and F. Tufaro. 1991. Examinarion of conditions affecting the efficiency of HVS- I amplicon packiiging. J Virol Methods. 52:2 19-29.

Yazaki, T., H. J. hlanz, S. D. Rabkin, and R. L. Martuza. 1995. Trcatment of human malignant meningiomas by G207. a replication- cornpetsnt multimutated herpes simplex virus 1. Cancer Res. 55:4752-6.

York, 1. A., C. Roop, D. W. Andrews, S. R. Riddeli. F. L. Graham, and D. C. Johnson. 1994. A cytosolic hrrpcis simplex virus protein inhibiis antigrn presentlition to CD8+ T lymphocytes. Cell. 775-5-35.

Zhang, X., H. O'Shea, C. Entwisle, M. Boursnell, S. Efstathiou. and S. Inglis. 1998. An efficient selection system for packaging herpes simplrn virus amplicons. Journal of General Virology. 79: 125- 1 3 1 .

5 0

CHAPTER 2: An Enhanced Packaging Systern for Helper-

Dependent Herpes Simplex Virus Vectors

Rcprinrcd from J of Virol. 72:7 137-7 1-13 i 1998)

';cite: .Additional details on Materials and Methods is provided in the Appendin.

TOM .A. STAVROPOCLOS ASD CRAIG A. STRATHDEE

Gcnc Theriipy and Molecular Virology Group. The John P.Robarts Rssearch Institutr.

London. Ontario. Canada N6A 5K8. and Depanment of Microbiology and Imrnunology.

Cniwrsip of Western Ontario. London. Ontario. Canada

INTRODUCTION

Herprs simplrx virus ( H S V ) is a neurotropic herpesvirus that can establish lifelonq

infcsiions in its human host through latent maintenance in the ganglia of sensop nèurons.

The virus is relatively well characterized. with a genomr of - 155 kb that is maintaincd as a

conçatemerized circular or linear rpisome in infected cells (27 ). Because of thsir wide host

range. r. ftïcisnt infection. long term persistence. capacity to accommodatr luge amounts of

iorcign DNA. and ability to deliver genes to post-mitotic cell types. considerable çffon hrts

bwn expendeci to develop gene transfer vectors that can exploit the naturril biology of HSV

i 1.10.19 ). Both hclper-dependent as well as helper-independent vrctors are currsntly in

u idc lise. Hrlprr-indrprndent HSV vectors contain the cornpletc viral gcnomti. but h l i w

dclctions in one or more essential viral genes. These genomes çan bc used lis a vector to

c~pi.c\s man! cDNXs by replacing other non-rsscntial sciqurnccs i 1 7 1. Hrlper-independent

H S V \ u ~ o r s thus have a replication-drfectivr viral phenut) pe. and can esttiblish a

productive infection only on complementing ce11 lincs that express the corresponding

deleted virai genc ( 5 ) . The major problem associated with this class of vectors in general is

ihat thcre is leaky or inappropriate expression of some of the immediate rarly and early

HSV senes in non-complementing ceIl types. which ultimately results in death of the

infected cell even though no viral replication has occurred ( 14-16.42). Such cytotoxicity

can bt: cornpletely eliminated when al1 of the irnmcdiate carly HSV genss are deleteri. but

ttiis results in w y low lrvrls of rransduced reporter gene expression < 28.29). Thus cn tp

into the latent cycle may be mandatory for sene expression and maintenance of

hulpcr-independent HSV vectors. effectively limiting their use to neuronal crll types. Even

W. the factors controlling the complete latency-associated shut down of viral gsnc

expression have not been clearly defined for HSV. and limited progress has bern made in

bypassing this shutdown to facilitate long t e m transduced gene expression ( 18.35.36).

Hrlper-dependent HSV vectors (commonly known as HSV amplicons 1 çontain

only the cis rlements required for HSV replication and packaging. the ori5 and poc

dements. respectively (8.34)(Fig. 2. IB) . Because the vector backbone is only a small

fraction of the size of the HSV genome. usually <IO%. HSV amplicons have the potential

io express a large number of genes or cDNAs. .Moreover. sincr they contain no

vir-al-ençoded grncs. these vectors have the potential to provide Ions tsrm gens expression

i n trciii\ducrd crlls by obviating the latency-associatrd shutdown of HSV srnr expression.

and cire thus well positionrd to takr full advantage of the ability of HSV to infect virruall)

cm- ! type of ccll (7.8.1 1 ) . The first practical systrm for HSV amplicons utilizrd an

ICP-l-delctcd. replication-defective HSV as a hrlper virus to providr the necessnry viral

rcplication and prickasing functions ( 9 ) . In this system. the amplicon wctor i trandectrd

inio an ICP-I- complementing ceIl line. which is then super-infccted with the helper virus

iind the resulting infectsous particles harvested following growth of the virus. Although this

~ipproach can yield titres of recombinant virus approaching 109 pfulml. unlrss there is ti

mcçhanism in place to provide for the selective replication and prickaging o f the iimplicon

wcror w e r that of the helper virus. the transducing lysatrs producrd arc hswily

contnmincttcd with helper virus (9.10). Even when selrctivr replication and packqinr of

the amplicon vrctor is provided. the helper virus represents IO-25% of the total yield of

1 i r i i h producrd i 25 .U) . Similar to the situation with hrlprr-indrpendçnt wçtors. this

he lper virus contamination leiids to significant delivery -usociatrd cy totoxicity in

iransduced cells. Although this problem c m potentially be eliminated through the use of

Icss cytotoxic forms of HSV to package amplicon vectors. such mutants typically grow

much more slowly and the yield of packaged amplicon is correspondingly lower < 35) .

h second generation packaging system was recently developed for

hrlper-dependent HSV vectors that yields transducing lysates free of hrlprr virus

contamirution. making i t the packaging system of choice for HSV amplicons < 71. The

systrm is centered around a set of five overlapping HSV-1 cosmid clones that together

encode the complete wild-type viral genome (1)(Fig. L I A ) . By specifically deletin? the piic

t.lcrnc.nt in the repetitive rr sequences of two of the cosmids. the cosmid set is rrndered

packdging-dcfcçtive. and whrn CO-transfectcd into rnümmalian crlls dong with an HSV

aniplicon wctor. only the latter is packaged into mature viral particles. The drlrtion of the

p i c elcnicnts nddrrsses the requiremrnt for selectivs piickaging of the vsctor ovrr the hrlper

viruh. H o w w r . it does not provide for any selective replication of the amplicon vrctor.

mi this may be why the overall yield of packaged vector particles is quite low. usually

< 1 Ob U./rIll.

In this report we describe the development of an enhancrd second gencration

pcickiiging system for helper-dependent HSV vectors. which we achirved by modifying

hotti thc packa~ing-defective hrlper virus and HSV arnplicon vector cornponrnts d ihe

iiirreni \)\[cm. We rnodified the helper virus by cloning the m i r e paçkaging-defrctivr

HSV-I senonit: ab a single infectious plamid in bacteria. Wr modifird the amplicon vector

h) incorporiiting the SV40 origin of replication. which acts as an HSV-independent

replicon to provide for the replicative expansion of the vcctor prior to piickaging into

i n f t x t i o u s HSV-I pcinicles. The combination of both modifications affords an Y-fold

incrtiw in the yield of packaged vector. and provides sufficient material in bench top scale

production to enable iri vivo studies for most applications.

MATERIALS AND METHODS

Cell lines and plasrnids. The BHK(TK-) ce11 line (hamster kidnry was

provideci by Paul Johnson and Theodore Friedman (UCSD. La JoIla. CA) ( 15). The SV40

T-antipen positive ZUT- 17 cell line (human embryonic kidney w u prot ided by Doug Bell

i Cniversity of Ottawa. Ottawa. ON) and David Baltimore (CalTech. Pasadena. CA). and

wcii, cultured with 400 ~ g / m l Geneticin (24). The Vrro ce11 line (African green monksy

h i d n q 1 LU^ provided by Dora Ho (Stanford University. Stanford. CA). Eiich cell linr w a

iultured in DMEM supplemented with 10% FCS. and was split 1 :4 ruçry 3-4 days usinp

\landmi mcthods. The PC ll c r l l line (rat adrenal pheochromocytoma~ uas cultured in

Db1EM wpplrmentrd with 3% FCS and 5% horse serum. and was differentiated by adding

murinc nrrve growth factor to a final concentration of 50 nglml ( Harlan Bioproducts for

Sciencc Inc.. Madison. WT)(39).

The pCL4 I plasmid encoding the HSV- 1 ilhs sene was provided by Jarne.\ Sniilt.~

i Cnnmi ty of Alberta. Edmonton. AB) (31 ). The pu4UgaVori vector that containi the

HSV- I prc clemrnt wlts provided by Dora Ho (12). The bacterial artificial chromosome

i BACI cloning vector pBAClOSL as well iis the BAC hoht .;train E. coli HS996 uere

p r u w M b! Mttlvin Simon and Hiroaki Shi~uya iCaITrch. Padrn i i . Ch) (301. The

WSV- I cozmid set C was provided by Andrew Davison (MRC virolosy unic. Cniwrsit) of

Glris~ow. UK) and the modified cos6Aa and cos48Aa constructs as well as the pHSvlac

wctor were provided by Cornri Fraefel and Alfred Geller (Harvard. Cambridge. 414)

(-1.7). The latter was modified to include the minimal SV40 ori element by ligatinp the

BqII1-HiudIII fragment of the pGL3-Promoter vector (Prornega Inc. Madison. WI) into the

iiriiy lie BmtHI site to grnerate pHSVlacOri ( Fig. 1 B 1.

Construction of the targeting vector. The BAC vrctor wiis constructrd by

\ubcloning a cassette containing the HSV- I pcic element and the U-hgment of the bactenal

1mZ grne into the S d I site of pBAC 1 O8L as a Sd-XhoI fragment ( Fig. 2. I A 1. The HSV

p i c . ccihsettr. was constructed using the pcDNAI1 expression vrctor (Invitrogen Inc. La

Jolla. CA) in which the two iVsiI sites within the multiple cloning site were fused. the !Vwl

site. convertrd to SolI. and the TfiI site convened to PucI-XhoI using oligonucleotide

linkcrs. The HSV- I pac element from pa4BgaVori was ligated into the StrlI site as a

E r n - . Y h I fragment using oligonucleotide linkers. after which the Siin site was convertcd

ro SdI-BwiHI- PncI. The completed BAC construc t was subc loned be tween the two L n d

sites of pCLl1 as a linearized BamHI fragment. To accommodate the BAC. an

digonuclsotide linker was used to first convert the SrrraI sites of pUL4 to BczI~HI sites.

such that the BAC can be excised as an intact fragment. The targeting vector was linearized

by digestion with HiridIII prior to CO-transfection into BHK cells with the HSV l a cosmid

;it ~i rtitio of 1 5 .

Packaging of HSV amplicons. All plasmid. cosmid. and BAC DKX wax

prcpiired using a modified alkaline lysis protocol. followed by purification over a

proprietary ion exchange column according to rnanufacturrrs protocols c Qiagen [nc..

L'alcnciü. CA). The pBAC-HSV constnicts were funhrr trerited to remove contaminating

bncterial sndotoxin using a proprietary reagent (Qiagen Inc.. Valencia. CA>. and stored in

rniall riliquots at - W C to minimize sshearing of the DNA dur to repeated frerze-thnwing.

Thc HSV- I cosmids were digested with PocI and rr-puri fied by phrnol/chloroforrn

txtrxtion prior to trcinsfection. The pHSvlac vectors were packageil into inf tx i i i>u\

partidcs by CO-trlinsfection with either the HSV Aa cosmid set or pBhC-V2 at ratio of I :4

usin? ti proprirtary cationic liposome formulation according to rnanuficturers protocols

i Lipofr.ciXMINE. Life Technologies Inc.. Bethrsda. MD). Crlls were triinsfected with

2 ug DNA + 15 pl LipofectAiMINE in one well of a 6 well culture dish when the! recichrd

SO-90% confluence. Following transfection N.N'-hexamethylene-bis-actetamide

i HMBA>(Siyma Chernical Co.. St. Louis. MO) was added to a final concentration of 2 miVI

to srirnulrite imrnediate early viral gene expression (23). The media was changed and fresh

HMBA added 24 hours following transfection. and the cultures grown until evidence of rhr:

\.ira1 cytopathic rffect was noted (usually a funher 1-3 days ). Viral particlrs wrrr harvttsted

by scraping the cells and supernatant into a strrile tube. and lysing the crlls in two rapid

frerze-thaw cycles. Cellular debris was rernoved by centrifugation at 1000 x g for 10 min..

and the viral particles in the supernatant were aliquoted for s t o q e at -80'C i 1 1 ). The yirld

of infectious vector andor infectious virus was detennined by asay ing serial dilutions for

13-glilaçto.;idme activity using X-gal histochemistry ( 2 l ) or for plaque forming iictivity ( 1 I 1.

respcçtiwly. on Vero cclls.

.Analysis uf virai DNA. Infeçted cells from eizht Pl50 culture dishrs were

hanvstrd b) scraping and centrifugation at 1000 x ,y t'or 10 min. then resuspended in 8 ml

u i TE buffer ( IO m i Tris-HCI [pH 7.81. 100 mM EDTA) and lysrd aftrr ridding a Iùrther

8 ml of TE buffer supplemented with 2% Triton X-100 using a Dounce homogrnizrr.

Ccllular debris was removed by centrifugation at 2.000 x ,q for 10 minutes. and capsids

urre pelleteci by centrifugation i i t 17.000 x ,q for 90 min. The capsids wrre resuspendcd in

0 . 5 tnl TEN buffer ( 10 mM Tris-HCl [pH 7.8). 10 m M EDTA. 150 miM NaCl i . and the

\%-cil capsicis wcre rçrnoved by digestion wirh 2 mp/ml proteinasr K and 0.15 w lv SDS

wns t i litial c.onc.t.nrration~ at 6 0 C for 1 hour. followed by phenol extraction and dirilysis ri,

ihrrc chanses of TE buffer t 10 mk1 Tris-HC1 [pH 8.01. 1 mM EDTAi. For lisrition

iucticms. 100 ng of DNA was utilized in a 10 ui reaction voiume to promote

rc-cirçdarizlition. After incubation ovrrnight at I1'C. 5 pl of the rcriction was

clrctroporiired into cornpetent HS996 bacterial cells using standard protocols. For field

inwrsion gel rlectrophoresis. 100 ng of DNA was loaded onto a 1.09 agarose gel in O.jX

TBE (90 m M Tris-HCI [pH8.3]. 90 mM Borate. 2 mM EDTA). and the gel run for 20 hrs

at 200 V and I1'C. Forward pulse times were ramped from 0.9 sec to 6.3 sec with ;i

t;)ric ard to reverse pulse time riitio of 3: 1.

PCR amplifications. HSV-specific primers were targrted to the LrL40 genr

(Primer 10: A C C A T A G C C A A T C C A T G A C C ) and UL4' gene t Primer

-12: GTCGTC--4GGG.LAGMCTTGAGG). and were designrd to arnpiify acrohs the cntire LTr4l

g m e . BAC-specific primers were targeted to the Forward rrgion iPrimer

BF: T A T T G A C A T G T C G T C G T A A C C ) and reverse region (Primer

BR: ATGTCGGC kG;WTGCTT.ieTG) flanking the P d cassette. and in conjunction w ith

priniers 40 and 41. respectively. were designed to amplify across the sites of BAC

inregration into the HSV- 1 UL41 gene. A high processivity Ttrq polymrrlise cocktail

i Espand Long Template PCR System. Boehringer Mannheim GmbH. Gemany ) was used

tOr al1 rextions. The annealing temperature for al1 primer pairs was 60'C. with 100 ng of

BAC tcmpiate DNA or 30 ng of cosmid templatr DNA in slich rraction. Dirnethyl

rulphoside wiis included to a final concentration of 10% in thosr rractions that involwd

curension across the GC rich HSV- 1 pac rlement. but orherwise the thermal profile and dl

othsr parameters for amplification were accordine to rnanufücturers recommcndations. PCR

product~ w r e rinalyzed by agarose sel rlectrophoresis using standard metholis.

RESULTS

Cloning strategy. The developmrnt of a packaging system for helprr-dependent

HSV vectors that could provide lysates that are free of contaminating helper virus was a

sipificrint brerikthrough in terms of facilitliting rhe efficient. non-toxic delivcry of

infectiou vector particles (7). However. there are a number of limitation', inhsrçnt to thi\

\!\teni that make it technically demanding to use. and that limit rhr ultirnatr yirld of

packagsd amplicon vector: ( i ) pückiiging requires the transfection of five overlappin_o

HSV- 1 cosmid clones. which must recombine to f o m a functionril HSV- I grnornr prior to

replication: i i i ) some of the cosmid clones are unstablr when propagiitrd in bacteria. and the

prepürcition of high quality DNA for transfections is therefore somrw hat laborious: and ( iii , trcinsfection sfficiencies approaching 100% are required in order to manimize yirld of the

plickaged ampiicon vector. We hypothesized that it should be possible to surmount thesr

limitations by cloning the entire packaging-defective HSV-1 genome as a single plasmid

D S A using a bacterial artificiai chromosome (BAC) cloning vector (30). This would

proïidr tttchnically simple and much more efficient method of reconstituting the helper

\.;rus in the packaging cell. The BAC vector was developed in response to the nerd to

stably propagate large zenomic DNA fragments in bactena. and thus it is likely that the

cloned HSV- 1 gemme will be more stable as a BAC than in the presently usrd cosmids.

The approach we pursued was to reverse rngineer a single HSV-BAC clone trom

iht. t'i\,c. original HSV- 1 cosmid clones that have the puc elrmrnts already delrtsd. and is

h a d iin the ability of the HSV- 1 cosmid set to genrrate a functional viral genornr through

reconibinütion in mammalian cells (1). Sincr it has brrn cistablished thnt only one p r c .

clcnicrit is rcquired for packaging of the HSV-1 senome (7 ) . and that p<ic.elements retain

thcir function whrn placed at an alternative locus (3.32). we inferred that i t should be

possible to reconstitute a packüging-proficient HSV- I gcnomr from the Aa cosrnid set by

prwiding a suitübls pcic eiement. Our strategy was to generate a functional virus from the

HSV Aa cosrnid set by targering a BAC vector contciining ;i single pot element to ri non-

c\wntiül HSV- I grnc through homologous recombination. The BAC vector friciiiratrh the

\uhscquent cloninp of the resulting recombinant viral genome in bricteria as a sin@

cunïtruct. The ptrc rlemrnt can then br removed from this çonstruçt Ui iirro to generate a

pncküging-defrctive HSV- 1 clone that ciin bi: usrd as a helper virus for the packüging of

HSV rimpl icon vectors.

Generation of an infectious HSV-1 clone in a BAC vector. WeseIected

the HSV- 1 LiL41 sene as the site of inteeration of the BAC vector. The Ur41 gene product

is a tegument protein that is incorporated into mature viral particles and is involved in the

degrridation of host cell mRNA to provide the viral host shutoff (vhs) îünction ( 7 1 i. Sincr

the ultirnarr use of the helper virus will be to provide efficient delivery of the packaged

HSV amplicon. wr rxprct that the elirnination of the potentially toxic vhs function from the

infrctious particlc shouid have a positive effcct on survival of the triinsduced cells. Wr

designcd (i CTL4 / targetin: vector that contained an HSV pcic cassette within a standard

FIG. 2 . I. Recombinant HSV- t constructs. ( A ) Structure of the UL-II-BAC tarpeting wcior. The top portion of the figure is a schematic representation of the HSV-1 grnome ~ind the sorresponding Sa cosmid set. comprised of cos6Aa. cos48Aa. cos 14. ços28. and o . Tlie LrL41 g ~ n e is loçatcd within ~ 0 ~ 5 6 . as shown rnlarged in the middlc portion of thc I'igiirc. Opcn rcading framrs are indicated by open arrows. The bottorn portion of the iÏgurc. indicrite\ rhc structure and orientation of the UL41-BAC targeting construct. including thc p i c cassette containing the HSV-I pcic elenient and the a-fragment uC the hxtc . r i l i l 1 u Z sene. The large crosses indicate the regions of homology betwsen the t q c t i ng 1 cctor and cos56 that will facilitate homologous recombination to generatr a plickaginc-proficienc recombinant virus. The relative location and orientation of the digonuclktidti primer pairs (40i-42. -Q+BR. -IO+BF) used for PCR are ris indiçated. PI.IIIICT\ 40 and 42 are located outsidr of the resion of homology with the UL4-BAC iqc.tirig construct. ( B) Structure of pHSvlac amplicon vector. The HSV- 1 oris and pic clcnient~ pro\ idc for helper-dependent packaging of the vector. Expression of the bacterial I ~ K Z reporter gene is controlled by the HSV-1 lE4 promoter and SV40 polyadenylation ripais. a h indicated. The SV40 ori element was cloned into the unique BnmHI site adjacent t u the HSV p u - element.

BAC cloning vector. and then transfected it into BHK cells along with the HSV Aa cosmid

set (Fig. 2.1A). The resulting recombinant HSV-BAC viral progeny were sxpandçd on

Vero cells to obtain sufficient quantities for purification of the viral genomiç D W . W t

clcctcd not to clone out individual viral recombinants ut this s r s r . but nither to sclcct b r

the tïitert recombinants in the population by growing [hem as a single pool. Sincr the

reconibinant HSV-BAC viral progeny have the BAC cloning vector intrgrated into the

LrL41 gcnr. these were cloned simply by re-circularizing the linrar viral genome and

r.lcctroporating the DNA into the BAC host strain HS996.

W s recovered threr indrpçndent clones. and chariicterized them by restriction

r.niionuclease fingerprinting with BamHI to determine which contained a cornplrtr and

intact HSV- I genome. Sincr all three had very similiir patterns (data not showni. we tïrst

detcrniined if any wrrr still infectious and able to genrrate plaques when triinsfcctd back

m i t , B H K cells. One of the clones was able to producr a functional virus in this asha'. and

tr;ir dcrignatrd pBAC-V 1. We iissessed the relative efficiency of plaque fomiation by the

pBAC-V 1 clone by cornparing it to rhat of the original. packaping proficirnt HSV- I cosmid

rct. The D'iA çonstructs were transfected into BHK cells in a sinsle wrll of a 5ix wrll

culture Jish. and after 1 days the viral particles were harvrsted and the total viral yield

dctrrrninrd by assaying serial dilutions for plaque formation on Vero crll monolayers.

Cnder these conditions. the pBAC-VI and HSV cosmid set yielded 5.0 x 101 and

6.0 s 102 pfu/ml. respçctively . Thus despite the fiict that it is deleted at the Ci4 1 locus and

contains only a single pctc element. the pBAC-VI clone producrd right-fold more virus

ptirticlcs whrn transfected into BHK cells than did the HSV cosmid set. which contains an

intact viral senorne that includes two pac elernents. This result substiintilitrs our initial

cxpectation that an intact viral grnornk fragment should bt: intrinsically more sftlcisnt at

mwriting a functional virus than five overlapping genomic fragments. C

We used three independent analyses to determine if the pBAC-V 1 clone conrÿins the

full HSV-I grnome. First. we characterized the site of integration of the BAC vector into

thc HSV-1 senome using PCR amplification (Fig. 1.7). Amplification across the rntire

C'i41 locus usin: the 47+40 primer set yielded a 4.3 kb product in cos56 which

corresponds to the intact UL41 grne. and an 1 1.2 kb product in pBAC-V 1 . which

ii>rrcsponds to integration of the compiete BAC targeting construct within the Ur# I grne.

Thc pBAC-V7 clonr is a modification of pBAC-V I and will be discussed in the following

w t i o n . Furthermore. amplification across the integration junctions of the BAC targeting

constnict using the 10+BF and 42+BR primer sets yielded products of 1.2 kb and 3.0 kb.

i~c~p tx t i~~c ly . confirrning that the targeting construct correctly ioralirrd the BAC in the

intcndcd location within the UL41 sene. Second. we andyzed the s i x of the Pd-Jigehreil

pB.AC-V I donc iising field inwrsion sel elcçtrophoresis. and found it to bc identical tu that

d' tlic ~ t x a r n i c DNA of the original pooled HSV-BAC recombinant virus at - 1 % kb

i Fig. 2.3.4 1 . Third. we compüred the BontHI restriction rndonuclrase finserprint of the

clonr to that of the genomic DNA of the original pooled HSV-BAC recombinant virus

(Fis . 2.ZB 1. hlthough a few minor differences in the patterns wcre notrd. these are liksly

attributable to heterogenrity in the pooled virus sample. For example. note that the

fragments at 1 1 .O. 10.5. 9.0. and 8.5 kb in the pooled HSV-BAC viral DNA are present at

h d l the expeçted intensity. whereas in the pBAC-VI sample only the 10.5 and 9.0 kb

f r a y i c n t are presrnt. but in this case iit full intensity. These differences are dur to

rccombination in the repeat regions of the HSV- 1 genome that genrratr four dtrrnativs

isonicrs of the v i r i l DNA sample (3.76). Inversion of the unique sequencrs CL and Cs

relatiw to ccich other by recombination exchange in the diploid resions of the grnomr

cenerates a distinct pattern for each isomer in the population when analyzcd by restriction C

cndonuclcases. The BmzHI fingerprinting analysis confirms that there is equal

rrpresentation of al1 four isomers in the pooled viral DNA sample. and that only one of the

isomrrs is rçpresented in the pBAC-VI clone. The results of al1 thrre analyses are

FIG. 2.2. Iniegrition of the BAC vector into the HSV- 1 Ut-/ l gene. PCR products were ;c.ncr;iir.d from HSV cos56 and pBAC-VI and pBAC-VZ with the indicated primer pairs. mi \cparatcd ihrough a 0.84 agarose gel. The loss of 1.0 kb from the PCR products dcriwd t'rom pB AC-V7 is due to excision of the pczc cassette from this clone. The markrrr i i w i in Iiinc .LI is the I Kb Plus Ladder (Life Technologies tnc. Bethrsda. MD).

FIG. 2.3. Annlysis of the recombinant BAC clones. (A) Analysis of the recombinant and cloncd HSV- I constructs by field inversion gel electrophoresis. Purifird viral gcnornic DS.4 fi,oiii ihc poolcd HSV-BAC recombinants was analyzrd without furthrr proçeshing. ir licrc;i\ dic pBAC-V 1 and pBXC-V3 clones were linearized by digestion uith P d prior to

uenorne at Iiwding uii thc gel. Thc orrow indiclites the position of the linearized HSV- I , - 1 % hb. Thc i kb P d fragrnent corresponding to the pïrc cassette exciscd from pB.AC-V I I \ riut rcw1it.d in this anülysis. The markrr used in lane M ib the Midrange PFG Marker I i S c ~ r England BioLabs. Bevrrly. MA). with correspondinp. sizrs in kb tndicatrd down the d s ut' ihc figure. t B I Restriction endonuclease fingerprinting of the recombinant and clund HSV- I constnicts. DNA from the various constnicts was digrsted with BtrmHI and i - c w l ~ t x i by rlectrophoresis through a 0.6% agarose gel. In ordrr to provide adequate conti-cit for the diffrrent fragment sizes down the entire gel. the figure shown is a coiripo\itt. of two diffrrent photographie exposures. The closed arrows indicatr the 1 1 .O. 10.5. 9.0. and 8.5 k b fragments corresponding to the different isomers in the poolcd rumbinr in t HSV-BAC genornes. with the larger closed arrows indicating the 10.5 and W ) hb t'rapmcnts corresponding to the isomer cloned in the pBAC-VI and -V? constructs. Thc upcn arrow indicatrs the 7.5 kb fragment corresponding to the excised BAC vector in thc p B K - V I sample. and the white lines indicate changes in the size of this fragment in rhe pooled recombinant HSV-BAC and pBAC-VZ samples. In pBAC-VZ the BAC vcctor is rcduccd in s i x by I kb due to removal of the p«c cassette. and in the HSV-BAC sample the BAC Lcctor 15 reduçed in sizr by 0.2 kb due to cleavage of the recombinant viral gcnurnt: ~ l h i n thc p~ slernent. The marker used in lane M is the 1 Kb Plu3 Laddtrr tLik Tcshnologir\ Inc. Brthesda. iMD). with sizes of the corresponding bands indicritrd in kb cion II the bide of the figure.

consistent with the conclusion that the pBAC-V 1 clone contains an intact HSV- I genomr in

w hich the UL4 1 grnr has becn inactivated. Wr have also done similar analyses using

pB.AC-V I clones that have bern serially subcultured in bactrria an3 hiiw never reço~~ered

an! rearrangements (data not shown). suggesting that the HSV-I grnome is completcly

rtabilized in the BAC vector. Thus we proceeded to the nrxt step in drvrloping a hrlprr

L i n i frce piickliging system by rendering pBAC-V I packaging-drfectivr: rhrough drlrtion

of t hr $ingle p i c clçment present.

Ceneration of a packaging-defective HSV-1 clone. The BAC clonins

stratrgy we dcveloped was designed to facilitate the efficient removal of the single pic

clcment i n the pBAC-VI clone. The HSV ptrc cassette is flanked by PMI rrtrictiun

cniloniiclc~is~ sites. and since Ptrcl dors not cut anywhere rlse in thc BAC vccror in the

HSV-I gcnonie. the cassette can br: cxciscd by digestion with P d followsd by

rc-circularizcition of the construct. To facilitate the identification of PM-drleted clones in

bacteria. we inçorporritrd the a-fragment of the bricterial IrrcZ gene inio the original P d

wsct tc . The pBAC-V 1 clones that contain the cassette are blue on X-gd containin= plates.

wht'rtl;ls clones that have Iost the cassette are white. We recovered a number of such clones

using this simple screen. and characterized two of them with respect to their B m i H I

restriction endonuclease fingerprint to determine if the HSV- I genomc was intact. Excrpt

for 3 1 k b rrduction of the 7.5 kb BAC-derived fragment corresponding to the loss of the

HSY p i c cassette. both clones were identical to the parental pBAC-VI construct. The

B m i H I fingerprint of one of these. designated as pBAC-V2. is shown in Fig. 2-38.

Analyis of the Ptrcf-linearizrd clone using field inversion gel rlrctrophoresis çonfirms that

ii is the same size as pBAC-V 1 (Fig. 2.3A).

Wr further characterized pBAC-VZ using the same methodology described for ihe

parental @AC-V 1 clone. PCR amplification across the entire UL41 locus using the 4 2 4 0

primer set yielded a 10.2 kb product, and amplification across the integration junctions of

the BAC tar_oetin; construct using the Q+BR primer set yielded a 2.0 kb product for

p B K - V 1 (Fig. 2.7). The loss of 1.0 kb from the PCR products derived from pBXC-VI in

cornparison to pBAC-VI is consistent with the loss of the HSV pac cassette from thih

clone. In addition. we transfected pBAC-VZ into BHK cells to determine if i t was d l

infcçtiou?; and abIr to generate plaques. as described previously for the p B X - V I ab well

as !ht: HSV cosrnid set. As expected h m the combined data indicating that the pcic rlement

~ t a h deletcd frorn this clone. no infrctious HSV- I particles were producrd. These re\ult\

MC corihi\trnt wirh the conclusion that pBAC-V2 cootains a replication-comprrrnt but

pach;iging-defsctiw HSV-I grnome. and that this construçt clin br: u3r.d to package

mplicon wctors that will be free of hrlper virus contamination.

Packaging of an HSV amplicon vector into infectious particles. Sincr

the pxkaging-Jefeçtivr HSV Aa cosmid set can be usrd as a hrlprr virus to package HSV

anipliconh ( 7 ) . and the resulting cellular lysates remain free of helper virus contiiminütion.

i ve cvaluatttd the ability of the pBAC-V? clone to function in a similar rolr. Usin: the

prototypical pHSvlac amplicon vector (Fig. ?.IB) üs a substrate. pBAC-VI wlis able to

provide appropriate helprr functions and package the vector to a titer of 8.0 i( IW bfulml.

and N irh no detectable hrlper virus contrimination. In cornparison with rhe HSV l a comid

wt. which yieldrd a titer of 2.0 K 1W bWm1 in a parallel transfection. pBAC-VZ LW

toiir-iold more proficirnt at packaging the pHSvlac vector into infrctious particles. Wc

h a t e reprated this experiment müny times in a variety of cell linrs and with a number of

different HSV amplicon vectors. and although there is considerable variation in the titer

berwen experiments. pBAC-V2 consistently yields approximately four-fold more

pcickqed vector than does the HSV Aa cosmid set.

The BHK ce11 line was used in these experiments because thry routinely provide

transfection efficiencies surpassing 90% in Our hands. However. as drscribsd for the

cosniid packiging set. any cell line can be used for amplicon packaging ( 7 i. Ws have found

iimilür high transfection rfficiencies can be achieved using 293T cells (24). whereas other

Iliboratories utilize the Vero-hased 2-2 ce11 line (7.33). Regardless of the cell line used. we

have never observed any difference in transfection efficiency for rithrr the pBAC-V2 or

HSV Aü cosmid set whrn used as n helper virus (data not showni. i t is unlikely that the

iiicrriared nmplicon packaging efficiency of the former c m be attributed to Jifkrences in

iraiidtxi;ibility. ~l l thoqh given the geater technical difficultirs in prcpÿring DNX from the

HSV Aa cosrnid set this remains a possibility. A more likely intcrpretation of this data is

that thc yisld of packaged amplicon vector in rither system is dirrctly relateci to the

inlèciivity of the transfected helper virus DNA. The pBAC-V? construct is therrforr more

ci'ficicnt than the HSV Aü cosmid set because i t does not require any recombination evtnts

io Scncratr. the replication-competent viral genome that is required to package arnplicon

W C tors.

Wt: utilizrd PC12 cells to examine the toxicity of the infrctioua pHSVliic vector

pcirticlcs gcncrated by pBAC-V? helper virus genome in n post mitotic cells modrl. When

p o u n in media containing nerve growth factor. PCl? çrlls exit the cell cycle and

diffcrentiatc to rrsemblr sympathetic nrurons in miin- aspects of cellular physiolo_~y (39,.

WC cornparrd the plüting efficiency of packaged p H S v l a c vector on difkrentiated PC 12

cdls and Vero cells. Since the B-galactosidase transducing titres were identical. we

conclude thar the pBAC-VZ helper virus genome provides al1 of the necessiiry HSV-I

proteins for efficient infection of neuronal cell types. Moreover. the vector-transducrd crlls

show no drtectable signs of toxicity ai three days post-infection (Fig. 2.4.). In this

csperiment. the lowest dilution of packaged pHSvlac vector that was plated out resulted in

a multiplicity of infection of 0.S panicles/cells. such that the majority of transducrd PCI2

crlls u-crr targrred by a single infectious particle. Because the HSV-1 vhs function is

deletcd from the pBAC-Vî helper virus genome. we exprct that infectious HSV- I püniçles

zcnçrated will be tolrrated equally wcll at much higher multiplicitirs of infection. for C

FIG. 2.4. Trilnsduced reporter gcne expression in post-mitotic crlls. Differentiüted PC 12 i ~ l l . w r e infectrd ür a multiplicity of -0.8 in a single well of a P34 culture dish. and 7 ~h),\ I m r thc cclls wrre fixed and stained for the pHSVlüc-derived O-gitlactosidiisr reporter gcnc x r i v i t y uh ing X - p l histochcimistry. The closed and open arrows indicrite positive and i i q i i t i w htiiining çrlls. rrspectivrly. The boxed ara in (B) indicatrs the location of the field h i u n iit highcr mügnitkation in (A) .

cuample as cün occur in vivo during direct injection of packaged vector material into the

nianiinalinn central nrrvous system. We now have data using rat and hamster modsls using

a vririety of different amplicon vectors that support this premise (manuscript in

prcprirrition I .

.An alternative viral replicon enhances HSV amplicon vector

püçkaging. The inrfficirncy of first generarion HSV packasin? systrms is derivcd from

ihcir iniihility to providr for the selrctive replication and pnckaging of the amplicon vector.

Thc HSV çosrnid set and HSV-BAC packnging systems work i n part bcciiusr the.

~pecificnlly üddress the latter deficiency through the use of a pückaging-drfçctive hclprr

virus. Since they do not select for replication of the amplicon vector. we postulated that the

!,iclcl of packagrd amplicon vector may be limited by ineffective cornpetition with the helper

virus for HSV-specific replication fictors. If this is the caw. thrn a imple solution is to

\pt.cifically increase the copy number of the amplicon vrctor relative ro thlit of the helprr

i i r u . In essence insuring that subsequent replication is skewrd to fiivour the vector rather

tli;in ihc helper virus. Our approach wÿs to use an alternative. HSV-indrpendcnt repliçon to

iii'lord mch a cornpetitive advantagr to the amplicon. and thus incrrasr the ovrrnll yirld of

piickaged vcctor particles. It is well rstablished that the SV40 origin of replication can

facilitate the szlective amplification of vinually any plasmid DNA in cells that express the

corrcsponding SV40 T-antigen (40). Accordingly. we utilizrd the SV40 system to

tictermine if this would have a similar effect on amplicon piickaging.

The pHSvlac vector was modified to include the minimal elements of the wrll

char;icterizrd SV40 ori. which includes the basal SV40 early promoter but not the enhancrr

i Fip. 2.1 B I . We compared the efficiency of packaging of pHSvlac and the modifird

pHSVliicOri in BHK cells and 293T cells (Table 2.1). In the T-antigen positive 293T crll

line (7-1,. the yield of packaged amplicon vector is increased almost two-fold whrn the

\.ector contains the SV40 ori element. This increase is not sern in the T-antigen negativè

TABLE 2.1. Enhnnced Packaging of an HSV Amplicon Vector.

C'cctor CeIl Linea Vsctor Tite$ Virus Tite+

- - -

pHSVlac 293T 1.5 x 10-s c l 0

pHSVlxOri 293T 2.7 x 10J 4 0

pHSVlac BHK 2.4 .u 1 W < 1 O

pHSVlxOri BHK 1.8 x 10-r cl0

.\The pBAC-VI clone was used as the HSV-1 helper construct in al\ transfections. hSrricil IO fold dilutions of the lysates were plated out on a confluent Iawn of Vrro cells. and c r l l s wcre assayed for B-@actosidase activity after 2 days using X-gal histochrmistry. The data is siven in blue forming unitslml of lysate. iSeriül 10 fold dilutions of the lysates were plated out on a confluent lewn of Vrro çells. and plaques wcre counted aftrr 3 days. The data is given in plaque forrning unitdml of l> u t c . 44 titer ol' c I O retlccts the fact that no helper virus was detrcted in a 10-1 dilution of the l y t c . which was the lowrst dilution tested in this rxperiment.

BHK céll line. where pHSvlac vector is packaged more cfficicntly than the modifisd

pHSVllicOri. In independent tnnsient transfection raperirnents we determined that P-

zcilactosidase expression from pHSVlacOri increased four-fold compared to thüt of

pHSvlac by 48 hours post transfection in 293T cells. but is unchanged in BHK cells (data

n a shown). We interpreted this result to correspond to an incnasç in copy number of the

SV40 ori çontiiining plasmid (pHSVlacOri) in the ce11 following transfection. Thcirefore ive

conclitdc that the rnhancrd pückaging of pHSVlacOri in 293T cc l ls i b due to

-r-;intigcn-dcpcndent pre-replication of the vector prior. and that this relatii.cl> imall

incrcahc in wctor copy number rffectively overcomes the slight packaging disadvantagr of

pHSVlacOri observed in BHK cells. Since in othcr expression systrms vector copy

numbcr can be increased by several orders of magnitude using the SV40 ori (-10). this

b~iggcst\ th31 further increases in the packaging efficiency müy be attainable by increasing

thc rcplicative efficiency of the added replicon. for example by increasing the cictivity of the

SV40 ori by inçorporating the full SV40 çnhancer. or by incorporating a more efficient

\.ira1 replicon into the vector.

DISCUSSION

The Jeveloprnent of gene-based therapies is currently bein: widely pursurd. und in

iiclds as diverse as infectious disease. autoimmune disease. crirdiovascular diserise. and

neurodrpenerütive disease. The availability of an efficient gene delivery and expression

\>.stem is an essential enabling technology that unites al1 of these applications. and as a

result this hiis bçen an area of intense research in recent years. HSV-based vectors show

considerable promise in this regard because of their wide host range. efficient infection.

long term persistence. capacity to accommodate large amounts of foreign DNA. and ability

[o Jttlivcr genrs to post-mitotic ce11 types (5.10.13). After funher optimizütion of cultiirr

m d transfection conditions and only a modest upscaling of our methods. ive have

sucçecdsd in reproducibly obtaining titres of I O 7 transducing particieslml of lysate for a

wricry of different HSV amplicon vectors using the pBAC-V2 helper çonstruct. Thus the

H S V packiging system is now efficient and economical cnough to providc sufficienr

iiiiirerial for most 111 i i i w studies. and we anticipate that it will be possible to ratrnd thcse ro

inciudr. expression of a therapeuric genr in various animal rnodrls of Jiseasr. In this

regard. future developrnents will likely be in refinements to th<: vcctor bückbonr itsslf in

ordcr tu \tübilizr the HSV arnplicon and thus afford rxtended trünsducçd grnc expression.

Onc approxh that we and othen have taken is to incorporate an alternative viral replicon.

for r..tarnple the spisomal replicon of the Epstein-Barr virus (37.41 ) or the in t rp t ing

rcplicon frorn the adeno-rissociated virus (6). and i t is likrly that additional hybrid HSV

miplicon vrctors are already in developmenr in othrr laboriitorirs. Given the rccent

i i i i p ro~ cnicnts in hr lpçr-indepcndrnt HSV vectors that rliminütc al1 deliw-) -~~'iwci;~tr.ti

c>toro.ticity (78.29). it will br interesting to compare the relative sfficirncy of long term

iriimducecl grne expression from this piatform with thiit obtained usin2 the newrr hybrid

ampl icon vector systrms.

The ability of the SV40 ori rlement to increase the yield of pückagrd vrctor in our

puckaging system stands in contrat to data obtained using a fully functionul HSV helprr

1.h-us. where the incorporation of the SV40 ori has no effect on arnplicon packaging (13).

Thus thc ability of pre-replication to increase the y ield of packagcd vector müy be

conteat-sprcific. for example dependent upon either the use of a pückaging-dcficient helper

\.irus md/or a sprcifir HSV amplicon. For example. the pre-replication by the SV40 (wi

t.lc.iiient rntiy only be effective only under those conditions in which replication of the HSV

iielpsr virus (and thus the HSV amplicon) are suboptimal. as is the case whrn a

replication-defective or packaging deficient HSV helper virus is used. In support of this

view. we have now incorporaied the minimal SV40 ori element into othrr HSV amplicon

i,r.ctors that Our Iaboratory has engineered. and have found that it is more effective in thosr

wctors thai arc ptickaged relatively inefficiently in comparison to pHSVlac. and can

increasr. ihc yields of packiiged vector by as rnuch as I O-fold in such cases i manuscript in

prepariition i.

The stratrgy we developed for cloning the HSV-I genome as a single infectious

BAC should br applicable to other DNA viruses. Indeed. during the course of Our

cspcriinenr a rcmarkably similar approach w u described for the murinr cytomegrilovirus

iniCMVi. ;i herpesvirus related to HSV ( 2 2 ) . In this case the experimsntal soal was to

~lewlop ;i systern in which the mCMV genome could be manipulated in bacteria. such thar

t/ic phcntmpe of viral mutants çould be defined without the requirrmcnt for growrh in ;i

rti;iiiirnalicin cell that is a prerequisite in conventional mutagrnesis protocols. In such u

~ti-iiicg! i t i iniperatix that the clontxi viral genorne be stable in bscieria. such that the

incidence of unwanted or cryptic mutations in othrr viral genes is minimizrd sincc thest:

u i>uld çonfound interpretation of the resulting viral phrnotype. To support our conclusion

thai rhc HSV-1 senome is completely stable as a bacmid. the growth kinrtics of the

HSV-BAC virus and virus derived from the infectious pBAC-VI clone are identical.

bloreowr. we have never detected any rearrangemrnts in the pBAC-V2 clone followins

rerid plissage in bacteria. suggesting that the cloned HSV genome appears to sritistj this

critcri~~. Thu'i we rxprçt that ri similar mutagenesis schrmr to chat described for the clonrd

niCMV senorne c m also be appliçd to HSV-1. The fact that we were able to p w x t r a

piickiiging-dcficiçnt but otherwise completely functional viral genomr illustr~tes the power

of such an iipproach. since it is othenvise impossible to propagate such a recombinant virus

in mlimrndian ccils. Note. however. that the pBAC-V 1 and pBAC-VI clones drscribed in

this study are deleted for the pcic sequences as well as U r l I . and are thus not well suitrd

t'or most other studies.

REFERENCES

Breakefield, X. O., and N. A. DeLuca. 1991. Herpes simplex virus for sene delivery to neurons. New Biol. 3203- 18.

Chiocca, E. A., B. B. Choi, W. 2. Cai, Ne A. DeLuca, P. A. Schaffer. M. DiFiglia, X. O. Breakefield, and R. L. Martuza. 1990. Trcinsfer and expression of the lacZ gene in rat brain neurons mrdiated by hsrpes simplex virus mutants. New Biol. 2:739-46.

Chou, J.. and B. Roizman. 1985. Isomerization of herprs simpleu virus I ocnoine: identifictition of the cis-acting and recombinarion s i t r within the domain Gi' the ii scqurnce. CeII. 11:803- 1 1 .

Cunningham. C.. and A. J. Davison. 1993. X cosmid-based systrm for constnicting mutants of herpes simplex virus type 1. Virol. 197: 1 16- 124.

Fink, D. .J.. N. A. DeLuca, W. F. Goins, and J. C. Glorioso. 1996. Gcne transkr to neurons using hrrpes simplex virus-basecl vectors. Annu R r v Ncurosci. t9:265-87.

Fraefel, C., D. R. Jacoby, C. Lage, H. Hilderbrand, J. Y. Chou, F. W. Nt . X. O. Breakefield, and J e A. Ma.jzoub. 1997. Gene transfer into hrpiitocytes mediated by helper virus-free H S ~ I A A V hybrid vectors. Mol Med. 3:s 13- 25.

Fraefel, C.. S. Song, F. Lim, P. Lang, L. Yu, Y. Wang, P. Wild. and A. 1. Geller. 1996. Helper virus-frer transfer of herpes simplrx virus type 1 pltismid vectors into neural cells. J Virol. 70:7 190-7.

Geller, A . I., and X. O. Breakefield. 1988. A dekctive HSV-1 vector expresses Escherichia coli beta-galactosidose in cultured pcriphrral neurons. Science. 241: 1667-9.

Geller, A. I., K. Keyomarsi, J e Bryan, and A. B. Pardee. 1990. i \n efficient deletion mutant packaging system for dekctivr herpss simplca virus wctors: potential applications to human gene therapy and neuronal physiology. Proc Natl Acad Sci U S A. 87:8950-4.

Glorioso, J e C., W. F. Goins, De Herpes simplex virus vectors and gene 87.

Ho. D. 1994. Amplicon-based herpes 43:191-210.

Ho, D. Y., T. C. Savdarn, S. L.

J. Fink, and N. A. DeLuca. 1994. transfer to brain. Dev Biol Stand. 82:79-

simplex virus vectors. Meth. Ccll Biol.

Fink, M. S. Lawrence, and R. M. Sapolsky. 1993. ~efectfve herpes simpiex virus vecrors expressing the rat brain ducose transporter protect cultured neurons from necrotîc insults. J Nsurochrm. 8 5 : ~ - 5 0 .

Huard, J., W. F. Goins, and J. C. Glorioso. 1995. Herpes sirnpirx virus type I vector mediated gene transfer to muscle. Grne Ther. 2:355-97.

Johnson, P. A., A. Miyanohara, F. Levine. T. Cahill, and T. Friedmann. 1992. Cytotoxicity of a replication-defective mutant of herpcs ~irnples virus type 1. J Virol. 66:7951-65.

.Johnson, P. .A., M. J. Wang, and T. Friedmann. 1991. Irnprowd cell h m - 1 i1.d by the reduction of immediate-carly senr expression in replication- defective mutants of hsrpes simplcx virus type 1 but not by mutation of the virion host shutoff tùnction. J Virol. 685347-62.

Johnson. P. A., K. Yoshida, F. H. Gage. and T. Friedmann. 1992. Effects of grne transfer into cultured CNS neurons with a replication- drfeçtive herpcs simplex virus type 1 vector. Brain Res Mol Brain Res. 12:95- 102.

Krisliy. D. M., P. C. Marconi, T. Oligino, R. J. Rouse, D. J. Fink. and J. C. Glorioso. 1997. Rapid merhod for construction of recombinant HSV senc trrinsfer vectors. Gene Ther. 4: 1 120-5.

Lachmann, R. H., and S. Efstathiou. 1997. Utilization of the herpes sirnplrx vims type 1 latency-associated regulatory region to drive stable reporter gent. expression in the nervous systern. J Virol. 7 1 5 197-207.

Leih. D. A., and P. D. Olivo. 1993. Gsne delivrry to neuronh: i hrrpr. rimplcx vims the right tool for the job'? Bioessays. 15547-54.

Lim. F., D. Hartley, P. Starr. P. Lang, S. Song, L. Yu. Y. Wang, and A. 1. Geller. 1996. Generation of high-titer defective HSV- I vectors usin2 an IE 2 deletion mutant and quantitative study of expression in cultured cortical cells. Biotcchniques. 20:460-9.

kIacGregor. G. R.. A. E. Mogg, J. F. Burke, and C. T. Caskey. 1957. Histochemicai staining of clonal mammülian ceIl lines expressing E. coli brta galactosidase indicates heterogeneous expression of the bacterial gene. Somat Crll Mol Genet. 13253-65.

Messerle, M., 1. Crnkovic, W. Hammerschmidt, H. Ziegler, and C. H. Koszinowski. 1997. Cloning and mutagenesis of a herpesvirus grnome as an infectious bacterial rirtificial chromosome. Proc Nat1 Acad Sci C S A. 94: 14759-63.

Paterson, T., and R. D. Everett. 1990. A prominent serine-rich region in Vmw 175. the major transcriptional regulator protein of herpes simplex virus type 1 . is not essential for virus srowth in tissue culture. J Gen Virol. 71: 1775-83-

Pear. W. S., G. P. Nolan, M. L. Scott. and D. Baltimore. 1993. Production of high-titer helper-free retrovimses by transient trrinsfrction. Proc Yatl AcadSçi U S A. 90:8392-6.

Pechan, P. A., LM. Fotaki, R. L. Thompson, R. Dunn. M. Chase, E.

A. Chiocca, and X. O. Breakefield. 1996. A novrl 'piggyback' packaging system for herpes simplex vinis amplicon vecton. Hum Gene Ther. 72003- 13.

Roizman, B. 1979. The structure and isomerization of herpes sirnplex virus senomes. Csll. I6:48 1-94.

Roizman, B., A. B. Sears, and R. J . Whitley. 1996. Fields Virology. Third ed. Lippincott-Raven, New York.

Samaniego, L. A., L. Neiderhiser, and N. A. DeLuca. 199% Ptirsistence and expression of the herpes simplex virus genome in the absence of irnmrdiate-early proteins. I Virol. 72:3307-20.

Samaniego, L. A., N. Wu, and N. A. DeLuca. 1997. The herpes simplrx virus immedinte-rarly protein ICPO affects transcription frrom the viral gcnorne and inkcted-ceil survival in the absence of ICP4 and ICP27. J Virol. 71A6 14-23.

Shizuya, H., B . Birren, U. J . Kim, V. Mancino, T. Slepak. Y. Tachiiri, and M. Simon. 1997. Cloning and stable maintenance of 300- kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-büsed wctor. Proc Natl .sicad Sci LT S A. 89:8794-7.

Srnibert, C. A., D. C. Johnson, and J. R. Srniley. 1992. Identification and characterization of the virion-inducrd host shutoff product of herprs simplex vinis gene UL4 1 . J Gen Virol. 73:467-70.

Smiley, J. R., C. Lavery, and M. Howes. 1991. The herpes simplex virus type 1 (HSV- 1 ) a sequence serves lis a cleavag.e/packaging signal but doçs not drive rccombinational genome isomerization when it is inserted into the HSV-2 genorne. J Virol. 66:7505- IO.

Smith, 1. L., M. A. Hardwicke, and R. M. Sandri-Goldin. 1992. Evidrncr that the herpes simplex virus immediate rürly protein ICP27 acts posi- transcriptionally during infection to regulate gene expression. Virology. 186:74- 86.

Spaete. R. R., and N. Frenkel. 1985. The hrrprrs simplet v i rus limpiicon: analyses of çis-acting replication functions. Proc Nati Acad Sci L! S A. 82694-8.

Starr, P. A., F. Lim, F. D. Grant, L. Trask, P. Lang, L. Yu, and .A. 1. Geller. 1996. Long-term persistence of defective HSV-1 vectors in the rat brain is demonstrated by reactivation of vector gene expression. Grne Ther. 3:6 i 5- 33.

Stevens, J. G. 1989. Human herpesviruses: a consideration of the latent statct. Microbiol Rev. 53:3 18-32.

Strathdee, C. A. 1996. Development of a hybrid herpesvirus vector for gene expression in mammalian tissues.. p. Abstr. W3 1-8. 15th Ann. meeting Amer. Soc. for Virol.. London. Ont.

? Y . Strathdee, C. A. Unpublished data. .

39. Tischler, A. S., and L. A. Greene. 1978. Morphologie and cytochemicül properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth Factor. Lab Invest. 39:77-89.

O Tsui, L. C., M. L. Breitman, L. Siminovitch, and M. Buchwald. 1982. Persistence of freely replicating SV40 recombinant molecules carrying a seltictable marker in permissive simian cells. Cell. 30:499-508.

4 . Wang, S., and J. M. Vos. 1996. A hybrid herpesvirus infectiou.\ vector based on Epstein-Barr virus and herprs sirnplen virus type I for p e trrinsfer into humtin cclls in vitro and in vivo. J Virol. 70:5422-30.

42. Wu, Y., S. C. Watkins, P. A. Schaffer, and N. A. DeLuca. 1996. Prolanped grnr expression and ceIl surviviil alter infection by a herpes simplrx virus mutant defective in the imrnediate-early grnes encoding ICP4. ICP27. and ICP22. J Virol. 70:635S-69.

4 . Wu, K., Y. Leduc, M. Cvnader, and F. Tufaro. 1995. Exriminrition u t conditions affecting the efficieniy of HVS- 1 amplicon packaging. J Virol .Llcthods. 52:: 19-29.

44. Zhang, X., H. O'Shea, C. Entwisle, M. Boursnell, S. Efstathiou. and S. Inglis. 1998. An efficient selrction system for packaging herpes simplrx virus amplicons. Journal of Genrral Virology. 79: 125- 1 3 1.

CHAPTER 3: DISCUSSION

The development of gene-based therapies is currently being widely pursurd in

t'iclds as diverse ris infectious disease. autoimmune disease. cardiovascular disrrise. and

iiciii-odegcnerative disrasr (34). The wtiiiability of an efficient gens dclivrry and

cuprcs\ion \!.stem is an essential rnabling technology that unites al1 of thess applications.

m d ris ri result this has been an area of intense research in reçent yeiirs. Irnperarivr: to the

wcccss of most gene therapy strategies in the c h i c are the two sirinecnt requirements

u hich niust br met by the vector: i ) efficient transfrr of therapeutic genei s) to tarset tissues

;inci i i ) cidequate expression of these gene(s) to alter cellular mrtabolism (34). For most

gcnr therapy applications long term expression of the therapeutic genr is fundamental in

order to provide a physiological benefit to the patient. howsver. the duration and level of

c ~ p r c s s i o n is Iargrly determinrd by the transgenr itself and the discase studied. Onl) in

b! \rems where cellular death of transduced cells is desired. such as trratmrnt o i maligniint

iuniuurs. is transient expression sufîïcient. Currently the vector systems thar have provcd

~iseful in delivering genes to targrt cells includr vectors based on rrirovirusrs.

tidenoti-uses r ADV). adeno-usociated virus ( AAV). recombinant HSV viruses. and HSV

mipl içons.

Therc are numerous human genetic disorders for which no treatment is currently

awilable. h proportion of these diseases afflict the central and peripheriil ncrvous systems

and lead ro drbilitating outcomrs in patients. Some of the most cornmon syndromes

indiide: ncurodrgeneration disorders such as Alzheimer's and Piirkinson's disrase.

neuronal tumours. rnetabolic abnormalities. physiological defects in developmrnt. and

irnrnunological pathologies. Because of the advances made in the genetic andysis of thrsç

disordcrs and since no real medical treatment exists for these patients. ii genetic approach

has been rationalized as a potential therapy (8. 24, 3 4 . Despite the efforts of rnany

rcscarchers rhis type of genetic intervention has not been possible to date.

The brain is an extremely complrx organ and reprrsents probably the most difficult

t i \ h i i r . in the body in which expression of therapeutic products throuph genrric transkr con

be richitl\.ed ( 2 1 ) . Because of the three dimensional structure and division of the brain into

distinct functional regions. localized gene expression will probably be nrcessary for a

numbrr of neurological therapies. This is a overwhelming obstacle since neuronal cells

unnot bc rctrievrd from the body for enplantation without dire consequrncrs to the hralth

of thc patient. Thrrefore. an in vivo approach is rsquired for the trcatrnrnt of CNS

ahii«riiictlities. The majority of gene tr~nsfer methodologies rely on direct intrriparenchyrnill

irijeciiun because vascular drlivery is blocked by the prrsrnce of the blood-brain barrier

( B B B 1. .-\lthou_oh the BBB crin be transiently bypassed with the use of h y pzrosmotic

wlutiuii.r io iillow passage of highly polarizrd molecules. these merhods have becn met

wirh limitcd success. panicularly with the use of viral vrctors < 17). Thus. viral vector

dclivery and targeting of specific regions of the CNS dcpends on effective sterrotactic

injcçrion protocols that can accurately administrr and maximize vcctor diffusion.

Additionrilly. the physiological conditions that confront expression of therapeutic products

in the C'IS by viral vectors include: i ) the availability of cellular receptors on the crll

w f a c e for viral infection. i i ) the mitotically inactive state of neurons and. i i i i the

inirii~inological r e q ~ o n x grneratrd by the hosr as a result of the presencr of t0reip

cintip-w and which will limit subsequcnt transgene persistcnce. Since neurons cire long

iivrd çrlls in the body. potential life long cures may be possible if a thrraprutic strategy can

overcornc rhcse obstacles and provide for stable gene expression.

Herpcsviruses have müny favourablr propenies that can be exploitrd as a thrrapy

thot rnsrts the aforementioned requirements of genetic intervention in the CNS. Firstly. the

viral genome naturally enters a latent state and persists without any physiological signs of

infection in neurons. Even in the face of a vigorous antiviral immune responss

herpesvimses can establish lifelong infections in the host through the maintenance of its

latent cyck (85). Additionally. the viral genome is maintained as an episome in infectrd

c d 1s. u hic h supports expression of therapeutic products independent of position r ffects

and aluo diminates the risk of insenional mutagenesis in the genome of the ce11 (10. 30.

3 I ). Thesr phenotypes have only been observed in neurons. and thus the nrrvous systrm

ha4 Iii~toriçlilly been targrted by HSV-based thcrapies. Fincilly. herpcsvirueh haw larst.

D S A genonies that çün accommodate a signitïcant ürnount of forrign grnrtic material.

Cnlikc . U V and retroviral vectors which restrict the amount of coding srqurncr available

10 therapeutic genrs. the potentiül coding çapacity of herpssviruses is siiffiçient to

cncunipass even the rnost complex thrrüpeutic expression strategics. Furrhermorr.

plaamids that contain the minimal viral replicon çan be cftïciently packaged inro infcctious

pxticles as concaternen. such that manipulating the vector senome can be cüsily done by

sr lindard recombinant techniques in bacteria (30). Becausr of their efficient infrctivity . long

tcrm persistence. capacity to accommodate large amounts of foreign DNA. and ability to

dçliver genes to post-mitotic cell types. herpesvimses shows considerable promix cix î

Jc l iw- and expression systrm for foreign genes in the nervous system (10. 74. 3 1 ) . The

tocus of this thcsis hos been on the utilization of one particular herpesvirus. herpes simplen

virus ty pr I i HSV). lu: a suitablc vector system for srne theripy.

There are two types of HSV vectors currently in wide use. Both types are Jesi_onsd

io takr ndvantage of the latent state of the virus such that the viral gsnome serves as

platforrn for foreign gene expression (8. 20). Sincr. the viral latent cycle only occurs in

neuronal c r l l types rhis lirnits the utility of recombinant HSV viruses to the nervous systrm.

The reason for this restriction is because the production of viral products which are not

compatible with cell viability cannot be actively suppressed by recombinant HSV viniscs in

uihsr d l types. In contrat. HSV amplicon vectors are able to stably transducc a number of

di ffrrent cc11 types wîrhout causing cytopathic rffects because they do not sncodr any viral

ccnei i 301. This attribute expands the application of HSV amplicons becausr thry can lilso

be usrd as p e therapy vectors for non-neurological defects.

The stability and level of gene expression is critical to gene thcrapy npplicütionb.

For the nioit part production of thrrapeutic protrins in tarset çrlls is detrrrnined by

imii~criptional rrgulatory elrments adjacent to the transgens. These includr promotrr.

cnhitnccr. cind silencing elernents. as well as transcriptional stan and mrthylation sites. The

iii:ijor problrm üssociüted with the use of HSV vectors in the CNS is that sens expression

from the vector tends to br down-rrgulated over time (71. 36). This cffrcr is most iicutrly

obw rved with recombinant HSV viruses becausr the presencc of viral genes in the vrçtor

stimulates the latrncy -associated shutdown of gene expression by the hosr d l . Even

though most recombinant HSV viruses are replication-defectivr and çannot transactivote

;in! ~ i r c i l gsnrh in the first place. this shutdown inhibits any prolongrd transgenc

cxprc\ iun (30). Although still unclear. it has bren suggrsrrd that HSV amplicon3 bufier

froni (i himilar down-replation in expression. A potential solution for this problem

i n w l w \ the utilization of non-viral prornoter sequencrs. for example. the cellular

promoters that drive neuronal housrkerping genrs (tyrosine hydroirylasc. or

prcproenkrphalin) (35. 40. 41. 83. 94). or the incorporation of regulatory elernents likr

cnhançers and locus control regions (LCRs) (53). As further studies revcal the precisc

elernents in promoters which facilitate prolonged gene expression. it will be interesting to

\ce i f entirely synthrtic promoters can be designed that :ive prolonged transgene

espression to vary ing degrees.

The major limitation to the utility of HSV amplicons over recombinant HSV viruses

lies in the ability of vector packaging systems to senerate enough infectious particles for

efficient transgene drlivery in riw. Recombinant HSV viruses can be propagatrd to high-

titre by sirnply growing the virus in culture (20). In comparison. amplicon packaging

,-stems are much more complex because two vimses (vector and helper) must be

efficiently propagated in order to achieve a high yield of packaged vector (23.43.69. 102).

The) ottrn in~olve numerous serial passages such that the ratio of packliged vector to

hclpcr iiruh is optimal or. transfecting large helper viral _osnomes into the cells of the

packaging reciction (27. 84). Additionally. they drmand close monitoring uf the Isvelh

hr.1pr.r virus and revertant populations in the transducing lysates. especially sincr

purification methodologies do not exist thm can physically rernove this contamination from

wctor stocks. Because up to 109 infectious vector particles are required to adrquately test

;in unplicon üpproach for transgene delivery iri riva. the success of thrar vectors ultimatel!.

rlcpcnds on an enribling technology that can produce enough material fret of contüminatinij

hc1pt.r virus.

Sincc the iidvrnt of the cosmid pückaging systcm ( 2 2 ) . HSV amplicon vsçtors have

bccunic more widel y üccepted as a feasibk gene therapy vector. The prima- reclson for this

ch;ingc i n opinion is dur to the hclprr virus frer transducing lysatrs grnrrcitrd by thc

cosmid-biised packaging system. This characteristic removes most of the cellular toxicity of

the wctor Iysütes and has allowcd researchers to begin to Iinswer questions conceming the

tifficacy of HSV amplicon vectors iri vivo free from the effects of the helper virus. The

pi-oblem with the cosmid packaging system is grnerathg enough starting rnatrriiil to ripply

tliis tcchnolop rffectively. My thrsis is an attempt at increasing the pückqing rfficienc).

and th113 the +Id of piicküged vector in a transient transfrction-biwd protoçol.

Ti> address the problems inherent to the cosmid-based packaging system dwrlopçd

for HSV amplicons. 1 cloned the complete helper virus HSV genome as a single infectious

DX.4 molecule in ii bacmid vector (84). Therefore. reconstitutinp the helprr virus as an

intact molrcule in the packaging ceIl through at least five distinct recombination cvsnts

betwcen the five cosmids would not limit the efficiency of vector packaging. Howrver. the

p B K - V 2 construct which does not require any recombinational events only confers an

approximate 4-fold increase in titre (Table 3.1 ). and therefore the fragmentrd nature of the

cosniid helper DNA is probably not a significant factor that limits progression through the

iytic cycle. The srgmented genome of the cosmid set can efficiently express a11 the rrtois

viral factors required for replication. even though only a small proportion of the helper

\.irus DNA sver recombines to generate a complete genome. Since the helper virus is not

requircd to form functional virus in either system. the structure of the helper virus gsnome

i < not critical to amplicon packaging.

Bec~iuse the hrlper DNA must be transfected into a packaging ceil line. high

iimdixtion dficirncics are required to maximize the yicld of packased vector i n the

rcactim tT;ible 3.1 L I have found thai BHK and 293T çells can routincly provide

transfcction rfficirncies surpassing 90% in my studies. As in the caht. describeci for the

coirnid packaging set (22). any ceIl line permissive to HSV can be used for cimpliçon

pcickliging by the bxmid construct.

The deletion of the p c elements from the transfected genomes of the b x m i d and

cornids cnsures the transducing lysütes generatcd are rcliitively helper virus-free

i <.O001 5 of the total infectious particles) (Table 3.1 ). This strategy limits the cytotoxicit)

produccd in the türgei çells. however necessitates transfection-based procedures to delivrr

the helper virus DNA to the packaging c d . By rnaintaining the packaginpdefrctivr

phenoiype of rhc helper virus in the bacmid clone. cornpetition for viral packaging factors is

s k s w d in fiivour of the amplicon and not the helper virus.

The major difference between the two systems is the bacmid-based rnethod is less

trchniçally dernanding to use because only one plrisrnid requires amplification/purificcition

from bacteria. whereas the cosmid based system necessitates the purification of fivr

individual clones. Moreover. durin% bacterial propagation cosmids 14 and 28 have bren

hnown to undrrgo spontaneous rearrangement which does not occur when the HSV

senorne is prown as a bacmid. Great care must be taken to ensure the intrgrity of rhr

cosmid DNA during isolation by restriction mapping. however. this technique does not

TABLE 3.1. Cornparison of Helper Virus-Free HSV Amplicon Packaging Systrms

- -

HSV- 1 Hrlper Grnorne Con figuration

C'L-CI Gcns

Hclpcr DNA Delive-

Spccializcd Packaging Crll Linc

Y isld ot' Packrisi-d Vsctor

Hrlper Virus Contamination

- - - - - - - - - -

set of 5 ovrrlüpping cosmid sin_ole BAC clone clones (cos6~ri . c o s l 8 ~ a . i pBAC-VZ) COS 14, COS?&. cos56 i

transfec tion

No

transfection

Y0

\sparate (i srnaIl percentagr of rearranged DNA giving rise to nonfunctional helper virus.

Bccaiise the lsvel of rearranzed DNA contamination varies between cosmid DNA preps so

do the titres of packaged amplicon. yet since pBAC-V2 is completely stable in bacteria. the

hdper 1:iral DNA stocks are homogrneous and give much more consistent titres

i 84). Finally. since both the HSV cosmids and the pBAC-V? construct were drrived

(rom 3 standard Iaboratory strain of HSV (strain F) (16) which has become adapted to

sroutth on çultured cells. any packaging systems developed from such strains may contain

rriutations which mnke in vivo transgene delivsry inherently inrfficirnt. For example.

csprtxion of sorne surthce glycoprotein may be down-reguliitrd since thry art. not rcquired

th- p w h in culture. howcver. sorne ceIl types in vivo depends on thess rrçttptors for

infcciiori. Thercforc. i t would be intrresting to determine if a more virulent hslpcr virus

D M senome. such as a cloned HSV- 1 clinical isolatc. çould be used to irnprovr the titres

ui' packagcd vector.

Aside from the modest irnprovemrnts in vector titre and the lcssrning of rhr

tcchnical dernand required to package HSV amplicons. the yield of packagrd vector

obtüincd remains approximately 100 times less than what is required to accuratrly evaluate

the tninsduction eficiency of HSV amplicons in vivo. For the rnost part this thesis is

focuwd on the utilization of HSV vectors for srne thernpy. howcver. a brief backsround of

thc ottier viral vector technologies will be provided in ordrr to facilitate a direct cornpanson.

Rutr-oi*inisrs: Retroviral vectors based on the ~Moloney murinc lrukemia virus are a

popular rnrithod for deliverinz senes to target cells because they c m provide highly efficient

x n e rransfer and stable gene expression (61. 73). The stability of retroviral vectors is C

primarily due to the capability of the virus to integratr its genomc directly into the host

chromosomal DNA. Therefore. as a consequence of normal cellular division the vector is

mnintained in every cell that originates from this precursor. This relatively simple

mrchanisrn provides a suitable expression platform of therapeutic products. howrver the

Ic~*el and duration of transgene expression is susceptible to the position effects of the site of

intsgriition i 61. 73).

No viral genes are required to be expressed for stable integration of the vector

becausr 311 the requisite enzymes are contained within the virion particle itself ( 1 3 . What is

necessary however. are the cis elements essential to vector packaging which inçludr the

LTRI. paçkriging signal. and primer binding site. Thrrefore. the majorit) of the 7 kb

wding capncity of retroviruses can be usrd to express a single large gene or two smallrr

cDS.h (77) . Howevsr. this may br problematic to the utilizarion of retroviruses in

c«itiplcx therapeutic stratesies. as in the case of rnultifi~ctoriül diseasrs which require man!

diffcrenr genrs expressed. Furthermore. incorporation of additional irqurnccs which

i-cgiilütc thc level of transgene expression such as specialized promotrrs. enhançers. and

insulriting elcments rnay be restricted due to the size limitation of the vector genorne and its

cfficiençy of packaging into the virion capsid.

The pückaging protocols for retroviral vrctors are similür to the onrs uscd for HSV

mplicon vcctors. Sincr a helper virus is required to provide the structural çomponrnts of

thc \ irion. retroviral vectors are packaged in a hrlper-dependent manner t 49. 58 ). In this

iysteni two plasmids that encode al1 hctlper functions are cotrmsfected with the vector itself

into the packaging ce11 line. One plasrnid contains the ,qrg and pol genes which rncods the

capsid çomponrnts and reverse transcriptase respective1 y. and the othcr provides the

cnvelope proteins (erw gene). Current packaging systems are capable of genrrating up to

10' infectious particles free of any contaminating helper virus (49. 58). However. the

virions are inherently unstable and subsequently cannot be concentrated to high titre for in

i i i w use. A strategy frequently practiced to deal with this problem is pseudotyping the

retroviral virion with the rnvelope proteins of another unrelated virus in order to increasse

ihr hoht range and stabilize the vector virion. To do rhis the glycoprotrin G protcin frorn the

\-rsicuiar stomatitis virus is substituted for the retroviral mi.. sene in the packaging protocol

i 27. 77 1.

The major limitation to retroviral gene transfer methodologies in the nervous systrrn

\tcrn% from the fact that the virion cannot actively enter the nucleus iollowin~ infection

I 27 1. Thereiorr. integration of the vector into the genome of the ceIl is depsiident upon

cellular mitosis to dissolve the nuclear membrane and allow the vector grnome access to

Iiost chsomosomçs. A s a consequence of this requirement. non-dividing crll types such as

iitxiruiis cannot be stably transducçd by retroviral vectors even though thshr: crllh arc

pcrrnis.\iblt. to virus infection. Lentivims vectors based on the human immunodrficienc)

L i r u h ( H I W represrnt a more cornplen form of retrovirus which has rvolved mcchanisrns to

facilitate nuclrar membrane transport and can stably transduce both mitotically active and

post-mitotic ceIl types (-13 . 59. 65. 66). Similar virus psuedotyping strategies describd

h n c . I i a w been applied to lrntivirus vcçtors büsrd on the human and f r l inc

~itirn~~nodc.ficiency viruses (FIV) as well as the rquine infectious linernia virus < EIAV in

a r d u t« incrt'asc the host range of the vecior ( 5 . 38. 39.60. 7 1 >.

The intrgration of retroviral and lentiviral vectors into the genorne is upparently

ranciom. al though a higher frequency of integration has bren corrr latrd w i t h

tranm-iptionally active regions ("open" chrornatin structure). Therefore. thrse vectors c a r y

(t potcntid risk associatrd with the production of insertional mutation by disrupting the

expression of a normal gene or by activating an endogenous proto-onco_~enr in the

transduced cell. General estimates of this frequency are roughly 10-6 for rach triinsducrd

crll. Sincr N I viw gene tninsfer protocols require delivery of approximatèly IOi'J infectiouh

pnstiçles. inregrating gene thrrripy vectors are better suitrd to rx i?iw applications whrre

Lin!. niutational cvents can be identified and selected against prior to tissue transplantation

i 13. 90).

Drprndovinises: Unique to most rnammals. including humans. are a group of

animal viruses referred to as dependoviruses. Dependoviruses are defective members of the

parvovirus bmily and as their name suggests. completely dependent for thtir replication on

the prrsrnçr of another virus. usually adenoviruses or hzrpesvirusès ( 3 1. Most often hrlper

funçtion is provided by adenoviruses and thus dependoviruses are more comrnonly known

;ts the adsno-associated virus ( AAV) (4). AAV can undergo two distinct phases in its life

cycle. a replicütivr lytic phase and a quiescent latent phase. The lytic phase is contingent on

hrlper funcrions provided by a CO-infecting adenovirus and results in newly replicated AAV

genonies pac k q e d into infectious particies. In the absence

cycle muintains its grnome in a provirus state through the

clirimownie 19 ( 50. 5 l I. The latent cycle üppears to be

of adenovirus. the AAV latent

integration ai a unique site on

u means of wrvitril t'or . U V

bccauhe thcw latent senomes crin be rescued and stimulateci to re-enter the lytic phase by

~ ~ ~ h q u s n r udenoviral infection. AAV is compietcly non-pathogrnic und thrrefore.

cuniicla-iiblc effort has been made to exploit the biology of AAV lis a vrctor for genetiç

interwrition (63. 64).

The . U V virion contains either a positive or negaiive polarity single stranded DNA

gcnome < 3,. Both forms are infectious and packagcd into infectious particles during the

&tic phase of the viral life cycle. The stmctural components of the virion are encodrd by

the r ~ i p genrs. and gives rise to a single RNA transcript that is üitematiwly spliced to yirld

thrw ciipsid proteins VP- 1 . VP-2. and VP-3. These proteins are assembled into the virion

sapiid i n iipprosimately a 1 : I: 10 ratio respectively. and mediate the infectivity of the virus

6 1 .

The other ORF in the viral genomr cncodes the rep genrs. or nonstructural proteins

of the virion. i\AV DNA replication and integration into the host genome is îontrolled by

the four rep proteins and the inverted terminal repeats (ITRs) (3). Gene therapy vectors

b a x d on AAV biology have taken a relatively simple configuration. Plasmid vectors

çontain the cDNA of interest with its appropriate promoter. intron. rnhancer. and

polyadenylation signals. flanked by the ITRs elements of the viral genome ( 6 1. 63. 6-11.

Packciging methodologies involve CO-trünsfecting the AAV vector with a plasmid rncoding

the r t ~ p and clip genes into 293 cells. and then super-infecting the reaction with adenovirus

uhich provides helper function for A M gene expression. The typical y ield of packaged

wçtor is approximately 106 transducing units per millilitre and contüins contaminating ADV

helpsr \.irus. Sincr AAV is extremely stable. furthrr purification and concentration

protocol~ ciin yield up to I O 14 infrctious particles free of any contaminating ADV < 19. 72.

97 1. The relative easc in preparing sufficient material for grnr thrrapy applications is the

niiijor dwntage of AXV vectors.

AXV vectors can infect a widr range of mammalian çell types regardless of rnitotic

b ~ i t i i 5 and thus have potential to provide stable genr trmsfer through the maintenance of the

I;itcnt phase by integrating into the host chromosome. This provides a suitable gent.

expression plütform however. the integration mechanism is relatively inefficient.

Approximately 100 rimes more viral particles are required to stlibly transducr the same

nunibcr of cclls cornpared to other more efficient viruses k g . herpesvirusrsi. This i a

iiiajor liniitiition impingine on the utility of AAV vectors. since i t mandates the production

of h igh ly çonçrntrated vector prepmtions which in iurn may lead to the grneration of a

wçtor-spticific immune response following administration (9 1 1.

;lde~ioi*intses: Along with retrovirusrs. üdenovirusrs are the most widely used and

besi studird delivery systems for gçne-based therapies (28. 61) . Vectors bassd on the

adenmirus biology take advantage of its ability to infect a wide variety of both replicating

and non-rrplicating ceIl types. and as such. gene therapy protocols have been rationalized

for cancer treatments and hereditary disorders like cystic fibrosis ( 15. 76. 96). The

adenovirus used. Ad7 or Ad5. is only a minor human pathogen and not bern associated

u i t h mal ignnncirs ( S I ). Therefore. ADV vectors are considered relative1 y safe for hurnan

gcnr thrrapq applications. The major technical advantages of sdrnoviral vectors over most

othrr vtictor systems is the relative ease in generating high-titre vector stocks for direct in

i*ii.o use. Currently. efficient packaging protocols exist for both helper-independent and

hclper-dependent ADV vectors and can yield 1010 or more transducing units per millilitre of

pic kagèci wctor.

.\DV has a linear double stranded DNA genome of appronimately 36 kb ( 8 3 . It is

iairl\. cornpltu and as a result early grneration vsctors wcre çonfi_gured as

hc1pr.r-independent types. Helper-indeprndrnt ADV vecrors contain Jeletions in one or

more resulatory genes which control viral gene expression. The E 1 . E 2 . andior E 3

transucti\-litor genes are most frequently rernoved from the genorne in order to

;icconirnodate the cDNA of interest and minimize the ionicity of the vector (981. ADV

wctors of ihis type thus have a replication-defectivr phenotypr and are complrmentsd for

gron-th by sprcialized cell lines. for example. rhe first generation ADV vcctors have a

rlclcrion in rhe E l gtne and grow on 293 cells ( 2 . 2 5 ) . These vectors are relaiively

Jctoxiîïtxi and cm stably transducc non-complementing crlls in culture. However. therr is

Icaky espression tiom the remaining viral genrs and a significant amourit of viral protein is

y t h e s i z ç d in the cell (98) . Although this is not ri problern in ceIl culture.

hç. l pcr-independent ADV vectors do not stabl y transduce cells Ni vivo because viral prote in

5ynthcsis stimulates a potent antiviral immune response (47). Bccausr thesr proteins are

highly imrnunogenic and the activation of cytotonic T-lymphocytes leads to ceIl loss.

hdpcr-independent ADV vectors are not well suited as ü platform for prolon_ged senr

expression (54. 70). Therefore. the rnajority of gene therapy applications for hrlprr-

i ncleprndrn t ADV vcctors are directed towards cancer immunothcrapiss IV here viral genr

expression acts an adjuvant and enhances tumour killing.

A different iipproach in ADV vecior design was taken in the development of hrlprr-

dependent vrctor types. In this system only the packaging signal and replication orizins

a with the i invertrd terminal repeats ITRs) are incorporated into the vector backbone alon,

transgenc of interest. Because no viral genes are present these vectors are commonly

rekrred to 3s "gii tless" ADV vectors (6 1 ). This con figuration specificdlp addresses the

immunologically-based problems associated with helper-independent vectors and has bern

~~iççessfiil for the mort pan in achieving prolonged sene expression.

Early packaging systems were hamprred by high levels of contaniinating hrlper

\ . i r u and failed to provide sufficient packaged vector for in vivo use. Howrver. technical

ridvançcs in this procedure with the advent of packaging protocol that cmploys Cre-

inediatrd c.tcision of the piickaging signal to remove the helper virus from the trünsducing

I!\citc\. ha3 cciuscd ii resurgrnçe in helper-depcndrnt vectorology (61. 67 . SI i. ADV

ticlper-dcpendent vectors can now be grown CO high titre and purifird awny from any hrlpcr

L i r u h in iinsle strp using dçnsity gradient centrifugation (68). The major advantase

heiper-Jependent vectors possess over helper-independent onrs is at Ieut twicr as much

furcign D N A can be accornrnodated within the vrctor gsnome i S 1 ) . Sincr only ;i maIl

t'rxtion of the approximately 36 kb ADV genome is contained within the helper-dependent

vxtor. wen the mosr complex therapeutic strategy can exploit adenovirus delivcry for p n e

intertmtiun.

hdrnovirus vectors are maintained as a non-rsplicating cpisomr in transducrd c d l ~

i X 3 . Thrreforr. they are rapidly diluted out and lost from dividing crlls. Thih fiict

st'lèctiwly restriçts the use of ADV vectors to non-replicating celis types. to use in r-r virw

approaches wlirre drus srlection schemtis sustain vector-positive crlls. or to cases where

oniy triinsirnt gene expression is desired. Additionally. incorporation of loreign rpisomal

maintenance elernents into U V vectors is difficult because they not usually compatible

u i t h lincar grnomes.

3.1 HSV Vectors vs. AAV, ADV, and Retroviral Vectors

HSV vectors have many advantages over these other vector systerns which makrs

thrm an attractive choice for gene transfer protocols to the central nrrvous systrrn (8. 74.

45 1. Because HSV is a neurotropic virus it has naturally evolved more efficient ways to

transduce neurons in vivo. Thus. compared to predominately respiratory tract viruses. for

esample AAV and ADV. fewrr HSV vector particles are required to transduce the same

number of cells. In general, with the exception of a few cell types i predominatrly mature

muscle fibres ) the infectivity of herpesviruses approaches two orders of magnitude highcr

t h;iii . U V or ADV virions. Because of this difference in efficiency . highly concrntrlitd

. A . W and XDV vrctor stocks are necded which in turn may lead to the generation of

wcior-speçific immune responses following administration (32. 33. 47. 9 1 ) . In hrt . the

major problem inherent to ADV vector systems is the immunotonicity of the virion (54.

70 i. Sunicirous studics have reponed an acute toxicity in the CNS following direct injection

of ADV in the brain. and furthermore. immunologiclil responses drvrloped in the

rc\piratop traits of these animais cven without vector administration i 1 . 10. 19. 52. 55 i.

This effect çorrrlated with a decrease in gene expression and w u evident regardless of the

wctor titre administered. although. significantly more toxicity ocçurrrd at higher

rtiii l t ipl icities o f infection. The reason being a severe inHammatory rcaçtion rcsults in

high-titre neutrdizing antibodies and a CD8-positive responsc is provokrd in responsr to

i i r d iinti~cns in the animal (54. 70). Since recurrent challenge with riilcl tyw adenovirus is

ii conimon occurrence in the population. efficient genr delivery may require speciaiized

irnrnunosuppressive regimes. rspecially if redosing or mucosal delivery is involvrd i 32.

33). Potentially this problem may be limited by the use of alternative vector configuration

t hat prevent viral gene expression and antigen presentation. The "gutless" helper-de pendent

XDV vcctors are considered the most likely solution to this problem. In contrasr. such

potcnt antiviral responses are not genented in HSV vectors systcms because the latent cycle

of the ciru supports genome persistence by naturülly suppressing viral srnc expression.

Additionally. the virion proteins which mediate transgene delivery are relativrly well

tolsrrited and do not lead to cell loss.

.-\nothrr advantage of HSV vectors is their ability to accommodate Ixgr amounts of

foreign grnetic material in the virion itself. Recombinant HSV virusrs and HSV ampliconr

tiii~e a cciding capiicity of l5 and 110 kb respectively. which is sufficicnt for cvsn the mmost

conipleic thrraprutic stratrgies (S. 24). In comparison. the coding çapacity of AXV and

retroviral vectors is much smallrr. approximately 5 to 7 kb (27. 28. 50. 63). This sizr

limitation poses n signitkant problem to the utilization of AAV and retrovirus as delivery

+stems for rnultifactorial disordrrs or diseases where the therapeu tic gene itsel f cvcerds

t h i limit. Duchenne's muscular dystrophy is the prime example because the coding

w p m s e for the dystrophin gene is about 14 kb. which does not includr my promoters or

rcgullitory elsmrnts (9. 1 I . 46). Therefore. recombinant HSV viruses. HSV amplicons.

ancl "~utless" ADV vectors may be the oniy systems availliblr that could carry such Iür_oc

irmsgcncs and providr for a thrrapeutic benefi t.

The latcnr cycle of HSV providrs an rxcrllrnt platform for the expression of

tIicr;ipcuti~ sentis in neuronal tissues. In fiict. HSV vectors have shown long term genorniç

itability with gcnr expression lasting for greatcr that 16 months (44). . \ I I othrr vector

$!stems have not shown comparable expression profiles with respect to number of iells

and diiration of expression in the CNS. With regards to the other vrctor systems. a

conipiirlttiw study reponcd by Blomer et r d . between an ADV. AAV. retroviral vector

baseci on the Moloney murine Ieukemia virus (MLV). and an HIV-basrd lentivirus vector.

Iound that genr expression in the CNS of adult rats by the lentivirus vsctor w u the most

efficient i 5 1. Titrematchrd vector stocks were strreotacticall y injected into the strintum and

hippoçampus regions of adult rats and P-Ga1 x t i v i t y was accrssed for up to 24 werks. The

MLV wctor failed to label any neurons which correlates with its inability to successfully

triinsducc non-dividing ceIl types. Approximately 50. 65. and 90% of the striatal neurons

containcd ADV. AAV. and HIV vector genornes respectively. and thesr levrls remainrd

constant throughout the 6 week period. Only the XAV and HIV vectors showed stable

reporter grne expression for the duration of the study, although. the level detected with the

.\AV vector was significantly less. presumably due to a low integration efficiency for

. U V .

Drspite vector persistence shown by the ADV vector. gene expression rapidly

decrelied past 6 weeks. This result suggests that in the context of an ADV vector.

prolonged gene expression is subject to suppressive forces in the CNS. This phenotype is

siniillir to what is seen with helper-independent HSV vectors. and may be overcomr: by

uhing dtrmative promoters. for example the LAT promoters of HSV (LAPI or LXP?). or

riciirotiiii housekcrtping promoters to drive trlinsyrnr expression i 35. 40. 4 1. 44. S3. 94 i .

FiruIl>. \ince . U V and HIV-based vectors have been known to intrgratr. directly into ihc

gcnonic of célls. the rxtrnt of gens expression from thrse platforms rnay solely depend

upon transcriptional nctivity and not the direct loss of the transgene from the cell (6. 38. 65.

66). Although the integration frrqurncies of thesr vrctor types is unclear. i t will be

interesting to ser if they can approach the stable transduction rfficiency of HSV vectors in

ncurons.

3.2 Potential Improved HSV Amplicon Packaging Systems

Sincc both the cosmid and bacmid amplicon packaging systems rrly on transfrçting

cclls to drlivrr the helper virus. thry are innately inefficient at initiating a viral lytic cycle a h

nvne of the tcgumcnt proteins accompany the viral genome (27. 84). Thosc tegument

prorein5 serve to transactivatr irnrnediaie eariy gene expression and hencr. set the stage for

an efficient productive infection. Additionally. transfection-based mrthods crin never ensure

txq c r i l receives a helper virus genome. which limits the total number of cells undersoing

Iyric infection and reduces the corresponding titre of packaged amplicon vector. %y using

an infsctious virus as ihç helper virus the limitations in trmsfection are overcornr because

evrry ceil on the rnonolayer can be infected easily. and potentially used to package

~tmplicon vrctors. However. the problern associated with virus-based packaging systrms is

thlit helpcr viruses are packaging-competent and the transducing lysates are heavily

contaminated. Eliminaring this contamination was the primary reason why the cosmid

systsm w u developed and widely used. even though the level of pückaged vector is 1000

fold Itiss. One possible way to boost the titres is to combine the piickii_jing-drfrctive

pliciiot~ pe of the bacmid transfection system with the higher packaging efficient! ot an

iiii;.ctious virus. Much likr how I developed a packaginz-drfective HSV bacterial clone by

sccoiiibining a single pcic sequence surrounded by Pacl sites into the virus and then

\ubscquently delsting it by restriction digestion. an analogous virus-büsrd system may bc

ciipiiirrrcd such that the pccc elrment is drleted from the helper virus srnome using a site

yxcific recombinasr. In this system a packaging ce11 line that was previously transfectrd

uith an amplicon vector is infected by the helper virus. and used to grneratr infectious

pcti-ricles. The cc11 l inr d s o constitutively expresses a recombinasr which catalyses the

cscision uf the pic rlemrnt from the hrlper viral genomit when tt penetrates the nucieur.

Theref«rc. the majority of infectious panicles genrrated contüin ampliçon veçtors becciuse

the hclper virus senome h a been converted to a pückaging-defectivee phenotypc by the

scconibinrise. Similitr to the packaging system developed for hslprr-deprndrnt ADV vectors

i 67 i . this strategy can takr advantage of virus infectivity to increasr the packaging

effiçicncy of rimpl icon vectors. while maintaining the packaging-defectivr phenoty pe of the

bacmid sy stem. In addition. rnany more cells c m be used in packaging reactions compared

to the transfection-based bacmid method. and thus provide a convenient way to upscale

prodiiction. Hopefully this strategy can provide a significant increase in packaged vrctor

yield. while iilleviatin_g the cost and inconsistencies of bacmid transfection.

4.1 Alternative HSV Vectors

By upscaling the transkction protocol 10 foid. I have succreded in reproducibly

obtiiining titres of 107 transducing unitslml of lysate using rhr pBAC-VI helper construct.

Thus. the HSV packaging system is now efficient and economicd enough to provide

sufficirnt rnriterial for preliminary in r.ii.10 studies. and I anticipate that it will be possible to

r.str.nd thew ro includr expression of a therapeutic gene in various animal modcls of

diseiisc. In this regard. future devcloprnents will likrly lie in rrfinemrnts to the vrctor

baclhonc itself in order to stabilize the HSV amplicon and thus afford extcndçd tmnsductrd

gens rupression. One way is to incorporate an alternative viral replicon. for cxtirnple the

spisomal replicon of the Epstein-Barr virus (86. 931 or the integrating replicon from the

adrlno-asmciated virus < 2 1 . 37). and it is likely that additional hybrid HSV omplicon

wctars are alrrady in development in other laboratorirs.

A d V-HSV iwtors: Breakefirld and col leagues have constructrd a hybrid amplicon

wctor tliat contains the inverted terminal repeats (ITRs) and the rep senr of .\..W. in

ddiiion t u the minimal HSV replicon (oris and ptrc rlrments) ( 2 1 . 37 1 . This vtxtor

w n i h i n e ~ the inkctivity of the herprsvirus for transgenr drlivrry and the intrgration

tiinction of AAV for stable vector maintenance. The vector is configured such that the

transgsns cDNA and its rrgulatory components (promoters. introns. tinhancers.

polydrnylation signüls. etc.) are flankrd by rhe ITRs. The rrp gene. which is rcsponsible

for replication and integration. is encoded on the vector biickbonr outside this region. In

rhis systrrn. the AAV-HSV vector genome is delivered to the nucleus of a target cell

through HSV infection. and then the ITR-flanked transgentis are amplifird and inregrotcd

into 3 ipwific locus on human chromosome 19 i 50. 5 1 1. The goal of this watcgy is ro

irnpürt rtability to the transgene by cellular intrgration. such that long term p n e expression

is possible.

The advantases of HSV-AAV hybrid amplicons ovcr their individual viral

cuniponrnts are three fold. Firstly. the AAV çomponents provide stable intrgration of the

transgenr in mitotically active cells. cornpared to conventional HSV amplicons which are

eventually lost in subsequent cell divisions (2 1 ). This provides a suitable platforni for long

term genr expression. Second. the HSV elements afford a more efficient yet less toxic

mrans of delivery compared to AAV infection. and finally. the size restrictions placed on

the transgrns integration cassette by the AAV virion itself are elirninated because HSV clin

;iccommodate much more DNA. Although not fully tested. in cornparison to standard

;inipliçons. the AAV-HSV hybrid vectors have considerable potential in providing gene

t.uprt.ssion in vinually any mammalian tissue ( 14. 37).

EBV-HSV recrors: An alternative hybrid vector that our laboratory is interestrd in

iricorporcitrts the cpisomal maintenance elements of the Epstein-Barr virus ( EB V into J

coiiwntionlil HSV iirnplicon i 86). EBV is a lymphotropic human herpesvirus that causes

lifelung latetir infections in humans. and is rndernic to the global population with ovrr 959

o f aclult\ being seropositivc (42. 75). The virus has a vcry high tissue tropisrn. and clin

onlv infect and replicate in two cell types: a productive lytic infection occurs in epithelial

cclls. wherras a latent infection occurs in B lymphocytes. which rire triinsformed into

dividing blast cells as a result (74). Lymphoblasts can proliferate indefinitely in culture and

niultiple copies of the - 170 kb EBV pnome. which replicates episomally at low copy

numbcr and only once per ce11 cycle. The viral genes that control latent replication Lire

clsari) distinct from those contributing to cellular transformation. productive \ ira1

rcplicaiion. and ceIl lysis. Specifically. the virus expresses elevrn gçnes during Iittcncy.

t.ncornprtssing six nuclear antigens ( E B N A - 1 . -2 . - 3 A . - 3 B . -3C. and -LP). three

membrane proteins ( L M P - 1 . -?A. and -28). and two small. non-polyadenylated R N A

i EBER- 1. and -2 ) (56. 80). Of these. only EBN.4- i is required for çpisomal replication

and niaintcnancc of the vin1 genorne dunng latency.

The EBNA - 1 gene encodes a multifunctional nuclear phosphoprotein that binds to

cis sequences in the viral genome located in the oriP element (87. 89). The EBIW-I/ariP

system functions as the episomal origin of replication as weli as a nuclear matrix artachment

region for the EBV senome. and also acts as an enhancer elsrnent that facilitate\

trtin\activation of other EBV latency-associated genes (7 . 18). Bacterial plasmids that

contain EBlV.4- I and oriP are not limited to replication in lymphoblast or epithelial cells

i 88. 99. 10 i ). and this observation has been exploited in the development of a variety of

EBV-basrd vectors for cDNA and genomic DNA expression (16. 93. 95). For the most

part thew \mors s h m common characteristics. and contain al1 the clcrnrnts required for

cfficicnt srlection and maintenance in v i f m . Their replicativr tldelity is quite high. they can

wppur t stable gene expression in both mitotic and post-mitotic human cell types for

cutendt. J periods. and they are only rarely associated with genomr altering inregration

c w n i (95. 100). Because the virus uses the EBNA-l/oriP systern to persist indefinitrly NI

i-ii-o i 37 ,. this implirs chat EBV-based vectors will share similar characteristics.

EBV-HSV hybrid amplicons take advantage of the EBNA- i/oriP systrm to impan

p t o m i c stability to the vector in transduced cells. By incorporating these two elements into

the conwntionctl HSV amplicon. the vector can utilizr the infectivity of HSV and iis broacl

tropisni ils ii cfelivsry systern. and the genomic maintenance characteristic\ uf EBV for

\iiihlc scne expression ( 86.93 ). Therefore. EBV-HSV hybrid vectors have the potentid to

prol-ide for long term expression in a variety of tissues for genr-based thrirapics.

Given the recenr improvemrnts in recombinant HSV vectors that diminate al1

Jelivsry-associated cytotoxicity (78. 79). it will be interesting to compare the rrlaiivr

cfficiency of long tcrm transduced gene expression from this platfom with that obtainrd

using the nrwer hybrid amplicon vector systems. Regardless. in order to perform these

cuperimrnts using HSV amplicon technology . a more advanced packagine systern must be

dcwloped that can generate high-titre packaged vcctor stocks. Clearly the

trmskction-baïed pückaging protocois currently utilized are not sütisfactory. and thsreforc

HSV amplicons have not realized their full potential in gene therapy.

REFERENCES

I I .

Akli , S., C. Caillaud, E. Vigne, L. D. Stratford-Perricaudet. L. Poenaru, M. Perricaudet, A. Kahn, and M. R. Peschanski. 1993. Trünsfrr of ü foreign gene into the brain using adenovirus vectors. Nat Genet. 3:224-8.

Bautista, D. S., M. Hitt, J. McGrory, and F. L. Graham. 1991. Isolation and characterization of insertion mutants in EIA of adenovirus type 5 . Virology . 182578-96.

Berns, K. 1. 1996. Fields Virology. Third ed. Lippincott-Raven. Xrw York.

Berns, K. 1.. and R. M. Linden. 1995. The cryptic lire style of üdeno- wocirited virus. Bioessays. 17237-45.

Blomer, C., L. 'laldini, T. Kafri, D. Trono, 1. 31. Verma. and F. H. Gage. 1997. Highly efficient and sustained sene transfrr in adult nrurons with a Ientivirus vector. J Virol. 71:664 1-9.

Blorner, C., L. Naldini, 1. M. Verma, D. Trono, and F. H. Gage. 1996. Applications of sene thrrapy to the CNS. Hum Mol Genet. 5 Spec NO: 1397-404.

Bochliarev, A , J A. Barwell, R. A. Pfuetzner, W. Furey. Jr.. -4. M. Edwards, and L. Frappier. 1995. Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein EBNA 1 . Cell. 83:3946.

Breakefield, X. O., and N. A. DeLuca. 1991. Herprs simplex virus for zene del ivery ro neurons. New B iol. 3:203- 18. C

Brockdorff, N., G. S. Cross, J. S. Cavanna, E. M. Fisher. M. F. Lyon. K. E. Davies, and S. D. Brown. 1987. The mcippinp of ti CDS;\ from the human X-linked Duchenne muscular dystrophy gcnr to the moux X chromosome. Nature. 328: 166-5.

Caillaud, C., S. Akli, E. Vigne, A. Koulakoff, M. Perricaudet. L. Poenaru, .A. Kahn, and Y, Berwald-Netter. 1993. Adenoviral vector as a csne delivery system into cultured rat neuronal and =lia1 crlls. Eur J Neurosci. b

5: 1257-9 1 .

Clemens, P. R., and C. T. Caskey. 1994. Grnr therapy prospects for Duchenne muscular dystrophy. Eur Neurol. 34: 18 1-5.

Coffin, J. M. 1996. Fields Virology. Third ed. Lippincott-Raven. New York.

Cornetta, K., R. A. Morgan, and W. F. Anderson. 1991. Safety issues related to retroviral-mediated gene transfer in humans. Hum Gene Thrr. 25-14.

Costantini, L. C., D. R. Jacoby, S. Wang, C. Fraefel. X. O.

Breakefield, and 0. Isacson. 1999. Gene transfer to the nigrostriata1 systern by hybrid herpes simplex vindadeno-associated virus amplicon vectors. Hum Gene Ther. 10248 1-94.

Crystal, R. G., N. G. McElvaney, M. A. Rosenfeld, C. S. Chu, A. Mastrangeli, J. G. Hay, S. L. Brody, H. A. Jaffe, Y. T. Eissa, and C. Danel. 1994. Administration of an adrnovinis containing the human CFTR ÇDNX to the respiratory tract of individuals with cystic fibrosis. Nat Genet. 8:Q- 5 1 .

Cunningham, C., and A. J. Davison. 1993. A cosmid-based systrm for constructing mutants of herpes simplex virus type 1 . Virol. 197: 1 16- 124.

Doran. S. E., ;Y. D. Ren, A. L. Betz, M. A. Pagel. E. .A. Yeuwelt. B. J . Roessler, and B. L. Davidson. 1995. Gene expression from recombinant viral vectors in the central nervous system a k r blood-brun barrier Jimiption. Nrurosurgery. 36:963-70.

Edwards, A. M., A. Bochkarev, and L. Frappier. 1998. Origin DNA- binding proteins. Curr Opin Stnict Biol. 8:49-53.

Ferrari, F. K., X. Xiao, D. McCarty, and R. J. Samulski. 1997. New dwelopnients in the genrration of ~ d - f r r r . high-citer rAAV genr therapy vrctors. 'irit Med. 3: 1295-7.

Fink, D. J.. N. A. DeLuca, W. F. Goins, and J. C. Glorioso. 1996. Gcnc transfer to nrurons using herpes simplrx virus-based vectors. Annu R s v 'icurosci. 19265-87.

Fraefel. C., D. R. Jacoby, C. Lage, H. Hilderbrand, J . Y. Chou. F. W. Mt, ,Y. O. Breakefield, and J. A. Majzoub. 1997. Gene transfsr into hepütocytrs mediated by helper virus-free HSVIAAV hybrid vtçtors. Mol bled. 3:s 13-25.

Fraefef, C., S. Song, F. Lim, P. Lang, L. Yu, Y. Wang, P. Wild. and A. 1. Geller. 1996. Helper virus-free trmsfer of herpes simplen virus type 1 plrisrnid vectors into neural cells. J Virol. 70:7 190-7.

Geller, .A. I., K. Keyornarsi, J. Bryan, and A. B. Pardee. 1990. An efficient delrtion mutant packaging system for defective herpes simple x virus vrctors: potential applications to human gene therapy and neuronal physiology. Proc Nat1 Acad Sci U S A. 87:8950-4.

Glorioso, J. C., W. F. Goins, D. J. Fink, and 3. A. DeLuca. 1994. Hsrprs simplex virus vectors and gene transfer to brain. Dev Biol Siand. 8 2 7 9 - 57.

Graham. F. L., J. Smiley, W. C. Russell, and R. 'iairn. 1977. Characteristics of a human ce11 line transformed by DNA from human adrnovirus type 5. J Gen Virol. 3659-74

Groger, R. K., D. M. Morrow, and M. L. Tykocinski. 1989. Directional antisense and sensr cDNA cloning using Epstein-Barr virus rpisomal r..;pression vectors. Gene. 81:Xj-94.

Gunzburg, W. H., A. Fleuchaus, R. Saller, and B. Salmons. 1996. Retroviral vector targeting for gene therapy . Cytokines Mol Ther. 2: 177-84.

Gunzburg, W. H., and B. Salrnons. 1995. Virus vector design in gene thrrripy. Mol .Med Today. 1:4 10-7.

Haase, G., P. Kennel, B. Pettmann. E. Vigne. S. k . F. Revah. H. Schmalbruch, and A. Kahn. 1997. Gçne therapy of murinr motor neuron discase using adenoviral vectors for neurotrophic factors. Yat Mrd. 3:429-36.

Ho. D. 1994. Amplicon-based herpes simplex virus vectors. Meth. CeIl Biol. 43:191-210.

Huard, J., W. F. Coins, and J. C. Glorioso. 1993. Herpes simple?; virus ty pc 1 vector mediated sene transfer to muscle. Gent: Ther. 2:335-91.

Ilan. Y., G. Droguett, N. R. Chowdhury, Y. Li, K. Sengupta. N. R. Thummala. .A. Davidson, J. R. Chowdhury, and M. S. Horr i t z . 1997. Insertion of the adenoviral E3 region into a recombinant virai vector prevents ant ivirill humoral and cellular immune responses and permits longterm gcnr expression. Proç Xatl Acnd Sci CT S A. 94:387-91.

Ilan. Y., R. Prakash, A. Davidson, Jona. G. Droguett. M. S. Horwitz. N. R. Chowdhury, and J. R. Chowdhury. 1997. Oral tolrrization to adenoviral antigens permits long-term gcnr expression using recombinant adenoviral vectors. J Cl in Invest. 99: 10%- 106.

Jane, S. M., J. M. Cunningham, and E. F. Vanin. 1998. Vrctor drvrlopment: a major obstacle in human gene therapy. Ann Med. 30:J 13-5.

Jin. B. K., M. Belloni, B. Conti, H. J. Federoff, R. Star r , J. H. Son, H. Baker, and T. Hm Joh. 1996. Proionged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simple?; virus amplicon vector. Hum Gene Ther. 7:lO 15-24.

Johnson, P. A., K. Yoshida, F. H. Gage, and T. Friedmann. 1992. Effects of genr transfer into cultured CNS neurons with a replication- Jefective herpes simplex virus type 1 vector. Brain Res Mol Brain Rcs. 12:95- 101.

Johnston, K. M., D. Jacoby, P. A. Pechan, C. Fraefel, P. Borghesani, Dm Schuback, R. J. Dunn, F. 1. Smith, and Y. O. Breakefield. 1997. HSWAAV hybrid amplicon vectors rxtend transgenc expression in human glioma cells. Hum Grne Ther. 8:359-70.

Kafri, T., U. Blomer, D. A. Peterson, F. H. Gage, and 1. M. Verma. 1997. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet. 17:3 14-7.

Kafri, T., H. van Praag, L. Ouyang, F. H. Gage. and 1. $1. Verma. 1 999. .A packaging ce11 line for lentivims vectors. J Virol. 73576-84.

Kaplitt, M. G.. A. D. Kwong, S. P. Kleopoulos, C. V. Mobbs, S. D. Ra bkin, and D. W. Pfaff. 1994. Preproenkephalin promoter yields rezion- .;pccific and long-term expression in adult brain after direct in vivo sene transfer via a dcfectivc herpes simplex viral vector. Proc Natl Acad Sci U S A. 9123979-83.

Kaplitt, M. G., and D. W. Pfaff. 1996. Viral Vrctors for Gene Delivrry and Expression in the CNS. Methods. 10:343-50.

Kieff, E. 1 !W6. Fields Virology. Third ed. Lippincott-Raven. New York.

Kim. V. Y.. K. Mitrophanous, S. M. Kingsman. and 4. J . Kingsman. 199s. Minimal requirement for a Irntivirus vitçtor basrd on human iinniunodehçirncy virus type 1. I Virol. 72:8 1 1-6.

Lüchmann, R. H.. and S. Efstathiou. 1997. Utilization of the herprs si inple x virus type 1 Iatency -associated regulatory reg ion to drive stable reporter x n r expression in the nervous system. I Virol. 71:3 197-107. C

Leib, D. A., and P. D. Olivo. 1993. Genr delivery to neurons: is herpea simplrx virus the right tool for the job'? Bioessays. 15547-54.

Lev. A. A., C. C. Feener, L. M. Kunkel, and R. H. Brown. Jr. 1987. Expression of the Duchenne's muscular dystrophy gene in cultured muscle cells. J Biot Chem. 262: 158 17-20,

Lieber. A., C. Y. He, L. Meuse, C. Himeda. C. Wilson. and M. A. Kay. 199% Inhibition of NF-kappaB activation i n combination wi th bcl-2 txprcssion allows for persistence of Cirst-genrration adenovirus veçtors in the mouse liver. J Virol. 72:9367-77.

Lim. F., D. Hartley, P. Starr, P. Lang, S. Song, L. Yu, Y. Wang, and A. 1. Geller. 1996. Generation of high-titer defective HSV-1 vectors usine an IE 7 drletion mutant and quantitative study of expression in cultured cortical c d 1s. B iotrchniques. 20:460-9.

Lin, X. 1998. Construction of new retroviral producer cells from iidenoviral and re troviral vectors. Gene Ther. 5: 1 25 1 -8.

Linden, R. M., and K. 1. Berns. 1997. Site-specific integration by cideno- ossociated virus: a b a i s for a potential gene therapy vector. Gene Ther. J:4-5.

Linden, R. M., P. Ward, C. Giraud, E. Winocour, and K. 1. Berns. 1996. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci C S A. 93: 1 1288-94.

Lisovoski, F.. S. Akli, E. Peltekian, E. Vigne. G. Haase, M. Perricaudet, P. A. Dreyfus, A. Kahn, and M. Peschanski. 1997.

h l .

Phenotypic alteration of astrocytes induced by ciliary neurotrophic factor in the intact adult brain. As revealed by adenovirus-mediated gene transfer. J Nrurosci. 17:7338-36.

Makarova, O., G. Gorneva, F. Wu, V. Farutin, B. Villeponteau. L. Poliani, D. Fink, and M. Levine. 1996. Incorporation of nuclru- matrix ~ittxhrnent regions into the herpes simplex virus type 1 grnome dors not inducc long-term expression of a foreign genr during latcncy. Gene Ther. 3529-33.

McCay, R. D., B. L. Davidson, B. J. Roessler, G. B. Huffnagle. S. L. Janich, T. J. Laing, and R. H. Simon. 1995. Pulmonary inflammation induced by incomplete or inactivared adenoviral particles. Hum Genr Thrr. 6: 1553- 60.

4lcCoy. R. D.. B. L. Davidson, B. J. Roessler, G. B. Huffnagle. and R. H. Simon. 1995. Expression of human intrrlrukin- 1 receptor antagonist in rnousr lungs using ÿ. recombinant adenovims: effects on vector-induccd intlammation. Gene Ther. 2:437-42.

Middleton, T., T. A. Gahn, J. kl. Martin, and B. Sugden. 1991. Immortalizing genes of Epstein-Barr virus. Adv Virus Rrs. 40: 19-55,

Sliddleton, T., and B. Sugden. 1994. Retention of plaamid D N A in

miimmalicin çells is enhanced by binding of the Epstrin-Barr virus replication protcin EBNA 1 . J Virol. 68:4067-7 1 .

Sliller, A. D. 1990. Retrovirus packaginz cells. Hum Genr Ther. 15-14.

5Ii!oshi. H., W. Blomer, $1. Takahashi, F. H. Gage. and 1. M. Verma. 1998. Development of a self-inactivating lentivirus veçtor. J Virol. 72:s 150-7.

SIiyoshi, H., M. Takahashi, F. H. Gage, and 1. hl. Verma. 1997. Stable and efficient gene transfer into the retina using an HIV-based lrntiviral vector. Proc Natl Acad Sci U S A. 94: 103 19-23.

1 0 , M. A., M. Gu, S. Motzel, J. Zhao, J. Lin, Q. Su, H. Allen. L. Franlin, R. J. Parks, F. L. Graham, S. Kochanek, A. J. Bett, and C. T. Caskey. 1998. An adenoviral vector deleted for al1 viral coding sequences results in enhanced safety and extended expression of a leptin transgenr. Proc Natl Acad Sci U S A. 957864-7 1 .

'rlulligan, R. C. 1993. The basic science of gene thrrapy. Science. 260:926-32.

?+Iuzyczka, X. 1994. Adeno-associated virus ( A A V ) vectors: will the? work'! J Clin Inwst. 94: 135 1.

>Iuzyczka, N. 1992. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 158:97- 129.

Naldini, L., U. Blomer, F. H. Gage, D. Trono, and 1. M. Verma.

1996. Efficient transfer. integration. iind sustained long-term expression of the rranssrnr in adult rat brains injected with a lentivirai vector. Proc Nat1 Acad Sci C S A. 93: 1 1352-5.

Saldini, L., U. Blomer, P. Gallay, D. Or?, R. SIulligan, F. H. Gage. 1. 31. Verma, and D. Trono. 1996. In vivo genr drlivrry iind stable transduction of nondividin; cells by a lentiviral vector. Science. 272:163-7.

Parks. R. J., L. Chen. M. Anton, U. Sankar, hl. A. Rudnicki, and F. L. Graham. 1996. A helper-dependent adenovims vector system: removül of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A. 93: 13565-70.

Parks, R. J., and F. L. Graham. 1997. A helprr-dependent system for iidenovirus vector production hrlps define a lower limit for efficient D M püçknging. J Virol. 71:3193-5.

Pechan. P. A., M. Fotaki. R. L. Thompson, R. Dunn. Chase. E. .A. Chiocca, and X. O. Breakefield. 1996. A novel 'pisgyback' packaging \y.;tr.rn for herpes simplex virus amplicon vectors. Hum Grne Ther. 72003- 13.

Piedra. P. A., G. A. Poveda, B. Ramsey, K. McCoy, and P. W. Hiatt. 1998. Incidence and prevaience of neutralizing antibodies to the common iidenoviruses in children with cystic fibrosis: implication for gene therapy with ridenovirus vectors. Pediatrics. 101: 10 1 3-9.

Poeschla. E. M., F. Wong-Staal, and D. J. Looney. 1998. Efficienr transduction of nondividing human cells by feline immunodeficiency virus lentivirril wctors. Nat Med. 4:354-7.

Rabinowitz, J. E., and J. Samulski. 1998. Adeno-associatsd virus expression systems for gene transfer. Curr Opin Biotechnol. 9:470-5.

Ramani. K.. R. S. Bora, M. Kumar, S. K. Tyagi, and D. P. Sarkar. 1997. Yovel gene dclivcry to liver cells using engincsred virosomes. FEBS L m 404: 163-8.

Rickinson, A. B. 1998. Epstein-Barr virus in action in vivo. N h g 1 J bled. 338: 1 % 1-3.

Rickinson, A. B., and E. KieW. 1996. Fields Virology. Third cd. Lippincott-Raven. New York.

Rosenfeld, M. A., W. Siegfried, K. Yoshimura, K. Yoneyama. M. Fukayama, L. E. Stier, P. K. Paakko, P. Gilardi, L. D. Stratford- Perricaudet, M. Perricaudet, and et al. 199 1. Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epitheliurn in vivo. Science. 252:43 1-4.

Salmons, B., and W. H. Gunzburg. 1993. Targeting of retroviral vectors for gene therapy. Hum Gene Ther. 4: 1294 1.

Samaniego, L. A., L. Neiderhiser, and N. A . DeLuca. 1998. Persistence and expression of the herpes simplex virus genome in the absence of imrnridiate-early proteins. J Virol. 72:3307-20.

Samaniego, L. A., N. Wu, and N. A. DeLuca. 1997. The herpes simplen virus imrnediate-early protein ICPO affects transcription from the viral genome and inkcted-ceIl survival in the absence of ICP4 and ICP27. J Virol. 71:46 14-25.

Sample. C.. and E. Kieff. 1991. Moleculür buis for Epstein-Barr vi rus induced pathogrnrsis and disease. Springer Semin Immunopüthol. 13: 133-46.

Schiedner. G.. N. Morral, R. J. Parks, Y. Wu. S. C. Koopmans. C. Lüngston. F. L. Graham, A. L. Beaudet, and S. Kochanek. 1998. Genomiç DNA transfer with a high-capacity adenovirus vector results in improvrd in vivo gene expression and decreased toxicity. Nat Genet. 18: 180-3.

S henk, T. 1996. Fields Virolgy. Third ed. Lippincott-Raven. New York.

Song, S.. Y . Wang, S. Y. Bak, P. Lang, D. Ullrey, R. L. Neve, K. L4. O' Malley, and A. 1. Geller. 1997. An HSV- 1 vrctor çontiiining the rat iyrosinr hydroxylase promoter rnhances both long-term and ceil type-spscitïc enprcssion in the rnidbrain. J Neurochrm. 68: 1792-503.

Stavropoulos, T. A., and C. A. Strathdee. 1998. Anenhanced packagin_o systern for helper-dependent herpes simplex virus vectors. J Virol. 7 2 7 13743.

Stevens. J. G . 1989. Human herpesvinises: a consideration of the latent srarc. .tIicrobiol Rèv. 53:3 15-32.

Strathdee, C. A. 1996. Developmrnt of a hybrid herpesvirus vrctor for sene expression in mammalian tissues.. p. Abstr. W3 1-8. 15th Ann. Meeting Amer. Soc. for Virol.. London. Ont.

Su. W., T. Middleton, B. Sugden. and H. Echols. 1991. DNA looping between the origin of replication of Epstein-Barr virus and its rnhancrr site: stabilization of an origin cornplex with Epstein-Barr nuclear cintigen 1 . Proc Nat1 Acad Sci U S A. 88: 1 O8704i.

Sugden, B., K. Marsh, and J. Yates. 1985. A vector that repiicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein- Barr vitus. Mol Ce11 Biol. 5:4IO-3.

Summers, H., J. A. Barwell, R. A. Pfuetzner. A. M. Edwards, and L. Frappier. 1996. Cooperative asscmbly of EBNA 1 on the Epstein-Barr v i w 1 a t m origin of replication. J Virol. 70: 1228-3 1.

Temin. H. 51. 1990. Safety considerations in somatic gene therapy of human diserise with retrovirus vectors. Hum Gene Ther. 1: 1 1 1-23.

Terarnoto, S., J. S. Bartlett, D. McCarty, X. Xiao. R. J. Samulski,

APPENDIX

Bacterial Strains Used

Competent cells for transformation at the DNA subcloning Irvel.

Bacterial host for bacrnid iimplificlition and purification.

Bacterial Growth Media

Luria-Bertani i LB 1 Liauid Media

Antibiotic Section 12.5 p@ml chlorarnphenicol

Luriri-Bertani Agar Plates

Antibiotic Section 12.5 pgml chloramphenicol

F.I.G.E. Buffers

I ,Y TBE ninnine buffer

90 mM Tris 90 mM Borate

2 mM EDTA

Agarose Gel Electrophoresis

l X TAE Buffer

40 m M Tris-Acetate 10 m M EDTA 0.5 @ml rthidium bromide cidjust to pH 7.9

B w r H I fingerprinting gels were first run üt 100 V to run the sampies into the _or1 and then run O\ ernight at 60 V tor adaquate separation of the DNA. PCR gels were run at 150 V for 45 min. The DNA was visunlized and photographed by exposure to short wave UV light.

Viral D N A isolation Buffers

1 X Viral Cell Lvsis Buffet- (TE)

100 miM Tris 10 rnM EDTA itdjust to pH 7.5

1 X DNA Extraction Buffer (TEN1

1 50 mM Nacl I O rnM Tris 10 m M EDTA âdjust pH to 7.3

1 X Dialvsis Buffer (TE)

10 rnM Tris I mM EDTA adjust pH to 7.5

1 X Viral Cell Lvsis Buffer (TE) + Triton4 100

100 rnM Tris 10 mM EDTA 0.2% Triton-X 100 adjust to pH 7.5

I-gal Based Histochemical Assay

I . Aspirate media from cells and rinse once with PBS. i PBS = phosphate buffered saline: IZ m M NalHP04, 3 mM NaH2P04 pH 7.3.

150 rnM NaCl)

1 . Aspirate PBS and overlay cells wiih fixitive and incubate at 4'C for 5 min. t Fixitive = 2% parafomaldehyde/0.2erc gluteraldehyde. JO m M Na2HP04.

10 miM NaH2P04 ph 7.3)

3 . Aspirate fixative and rinse once with PBS.

4. Aspirate PBS and overlay X-Ga1 stain. i %Ga1 stain = 80 m M Na2HP04, 10 miM NaH2P04 pH 7.3. 1.3 mM iM_oCl2.

3 m M K3Fe(CN)6. 3 m M hFe(CN)6. and I m g m l X-Ga1

PCR Conditions

500 uhl dATP 300 uhl dTTP 500 uhI dCTP 500 p.Cl K T P 300 nk1 dounstream primer 700 n M upstream primer 30 n p cosmid or 100 ng HSV-BAC template DNA 2 3 u n m Taq DNA polymerase 5 pl PCR buffer IOX = 500 mM Tris-HCl. pH 9.2 (25'C).

160 m M (NH4)fi04.?3.5 mM MgC12. 20% ( v h ) DMSO. 1 YC ( v l v ) TweenGEO

Total reactionvolume is 50 pl.

t N drnciture trmplatr 2 min at 94C

I denaturrrtion rit W C for 15 s I OS { iinnraling at 60'C for 30 s

i elongatton at 68'C for 8 min

I denrituration ;it 94°C for 15 s 20.Y ( annaling at 60aC for 30 s

I riongation at 68°C for 8 min. + cycle elongation for 20 s for cach cycle

I X 7 niin rit 68:C

1 .Y 45 min at 30°C

.A Hybaid Ornni Gene thermocycler was used to amplify the DNA (Hybaid Ltd.. Tcddington. 4liddlrsex).

BAC DNA Purification

Evsn though the cloned HSV genome is stable in the BAC vector. we still use the same prccautions iissociated with the previous cosmid clones (ie. nevrr colony puri- ).

Sote thnt the pBAC-V2 construct is -160 kb in size. so to avoid shearin: always pipet purified DNA with a cut off piprt tip and never vortex. as we tïnd that linear DNX is > 100 fold lcss efficient üt packaging than supercoiled DNA.

I . Streak out on a fresh plate of pBAC-V2 from a frozen glycerol stock. and grow on a LB plus ll.jpg/ml chloramphenicol plate ovemight at 37'C.

2 . From this frrshly streaked plate inoculate 2.5 litres of LB plus 13.5pg/ml chloriimphenicol with a loopful of bacteria and grow ovtlrnighr at 37-C wi th shaking.

3 . Sinçe the virus genome is contained in a BAC backbone. and such present as a \ingle copy in bactrria. it is difficult to obtain Ilirp amounts of high quality DEYA i b r transfection. Wr isolate our pBAC-VZ DNA using the EndoFree Plüsmid Mrga Kit {Qiagen Inc.Vr\lencia.CA). Although other methods of isolation exist we obtain icproducibl<: packaging titers with this protocol. Following isolation resuaprnd the DN.4 pellet in 1.5 ml of Tris-HCI (pH 8.5 1. quantitate using 0D26i,/2ni) and store lit - 2 O T at a concentration of 0.7 pg/pI in small aliquots untill use to minimize degradation due to successive freeze thaw cycles. Our typical yieid from n 2.5 litre culture is 100 to 350 pg.

Amplicon Vector Packaging

The following 1s a modification of the LipokctAiMl'iE Plus RciigentTJl trünsfeçt ion protocol t Lifc Technologies Inc.. Bethesda. iMD) that we have found to brt the most efficient for packaging HSV amplicons vectors. Values are oiwn for one trcinsicction/pack;1ging reaction in a 60mm culture dish. To upscale. ci&er incrrabr: the iiumber of transfrctions (multiple plates). and/or use a laser culture dish. Note rhat you ma! have to re-optimize transfection conditions for the latter. Although packaging reactions can hc cürrird out in any cell line permissive to HSV- l . it is important to choosr one that clin be highly transkcted. Since in our hands wc can routinely get cipproxirnately 90% rriin\ft.ction of 293T or BHK-II cells using the LipofectAMINE protocol we utilize thesr crll linrs for vrctor pacckaging.

1 . Plate 293T or BHK tk- cells in a P60 culture dish (60 mm) the day before so rhat the cells are 90-95% confluent at the time of transfection. Note that 293T cells require 4 0 pgml Geneticin.

7 . Pi pet 150 p1 of serum free media into sach of two 5 ml tubes.

3 . With a cut off pipet tip. add 3.5 pg of pBAC-V2 and 0.5 pg of amplicon vector to one of the tubes. Avoid vortexing or pipetting up and down vigorously during mixine. Let stand at room temperature for 5 min to equilibratt DNA.

Add 10 pl of LipofectAMINE Plus Reagent slowly to the DNA solution whilr moving the tip around to avoid precipitating the DNA. Mix by senrie tlicking or inverting tube and let stand for 20 min at room temperature. If a precipitate forms. discard the tube and start over.

Adcl 15 pl of LipofectAiMINE lipid to the second tube containin? 230 pl scmm frer media and mix.

Combine thc two solutions by slowly adding the DNA to the LipofectAiLLINE lipid rube u m g a cut off pipette tip and while moving the tip around to avoid precipitating the DNA. ~Mix and let stand for 20 min at room temperature to alIoa. for complen formation. If a precipitate forms. discard the tube and stan ovcr.

Rrmove media from crlls and wash once with pre-warmed semm fret media. aspirate. and add I ml of semrn free media back to the ceils.

L i n g a cut off tip. add the DNALipofectAiMINE complexes to the crlls rvrnly and w i r l to distribute. Return to incubator for 3 10 5 hrs.

Add 2.5 ml of DMEM (Dulbecco's rnodified ragle medium) containing 20% FBS i feral calf serum) to the culture to stop the transfection rextion. and then rrturn to the incubator overnight. Wr also add HMBA to a final concentration of 2 m M to stimulate immediate early gene expression from pBAC-V2 ctt this point [HMBA = N.N '-httxamrthy ienr-bis-acetamide].

N e ~ t düy replace media with 5 ml DMEM plus 10% FBS and 7 m M HMBA. thcn retiirn to incubator for a further 2-3 days (unti i crlls show significant cytopiithis efkct 1.

Harvest pückagcd vector by scraping the crlls into a 15 ml tube. Wiish an): residual cells from the plate with 5 ml cold PBS. The lysate is kept on ice from this point onwards.

Centrifuge at 100 x ,y, 4 OC for 10 min. and aspirate all but -500 p1 of media.

Vortex to resuspend cells. then lyse by sonication to release the infectious particles t'rom the cellular membranes. 'lote: Sonication should always be done on ice and for 4 cycles of 30 s on followed by 30 s off.

14. Centrifuge at 1000 x g. 4 OC for 10 min to pellet debris. and collect supernatant.

Repcat. Store supernatant nt -70aC as a transducing lysate in suitable aliquots until use brcause rrpeatrd frrrze thaw cycles lowers the titer of packaged amplicon. Depending on the vrçtor being packaged. our typical yield for a single 60 mm culture dish ranges from Sz 105 to 5x 106 total transducing units.

PCBLISHED ABSTRACTS

Stavropoulos. T. A. and Strathdee. C.A. 1999. A Packaging Deficient Herpes Sîmplsv C ' i r u h for Hrlpsr-Dependsnt HSV Vcctors. 2nd Ann. Am. Soc. of Grne Thrrap!.. Wtlhhington. D.C. Abstrrict No. 50 i .

Sta\ropoulos. T. A. and Strathdrr. C.A. 1998. An Enhancçd P o c k q i n g Systsm for Helper-Dependent Herpes Simplex Virus Vrctors. I sr Ann. Am. Soc. of Grnc Thrrapy .. Scattic. W.\. Abstrrtct No. 58.

Mrirsh. D.R.. Weaver, L.C., Strathdee. C.A., Cassam. X.K.. Mabon. P.J.. S tavropoulos. T. A. and Dekaban. G.A. 1998. Neuronal Infection of Dorsal Root Ganglia in vivo and Spinal Cord Organotypic Slice Cultures with Herpes Sirnplex Virus Amplicon Vcctoi-S. I st Ann. Am. Soc. of Gene Therapy.. Seattle. WA. Abstract No. 39 1.

\,lar\h. D.R.. Strathdee. C.A.. Dekaban. GA.. Cassarn. A.K.. Stiivropoulos. T. A. and lL'cri\.t.r. L.C. 1998. HSV Amplicon Vectors Infect Neurons of Spinal Cord Organotypic Slicr Cultures and Dorsal Root Ganglia in vivo. Soc. of Neuroscience.. Los Angeles. CA. Ahstrrict No. 24:#675.1 1 .