targeting hsv amplicon vectors

Methods 33 (2004) 179–186

www.elsevier.com/locate/ymeth

Targeting HSV amplicon vectors

Paola Grandi,a Matthew Spear,b Xandra O. Breakefield,a and Samuel Wanga,*

a Departments of Neurology and Radiology, Massachusetts General Hospital and Neuroscience Program,

Harvard Medical School, Charlestown, MA 02129, USAb Radiation Oncology, UCSD Cancer Center, Gene Therapy Program, University of California San Diego, San Diego, CA 92103, USA

Abstract

Several techniques have been developed to deliver DNA directly into mammalian cells, spanning in difficulty from simple mixing

procedures to complex systems requiring expensive equipment. Viral vectors have proven able to deliver genes into mammalian cells

with high efficiency and low toxicity. In particular, herpes simplex virus type-1 (HSV-1) amplicon vectors are well suited for gene

transfer studies as they can infect many cell types, both non-dividing and dividing, have a large transgene capacity and are easy to

manipulate. For some applications, it may be desirable to target gene delivery to specific cell populations or to transduce normally

non-susceptible cells. This can be achieved by modifying one or more of the glycoproteins found in the viral envelope. Glycoprotein

C (gC) has a well-characterized heparan sulfate binding domain (HSBD) necessary for HSV binding to cells. Replacing this region

with unique ligands can result in less efficient binding to natural target cells and increase binding to cells which express receptors for

these ligands. A method to retarget amplicon vectors by replacing gC HSBD with a model ligand, the hexameric histidine-tag, is

described, as well as means to evaluate the binding of modified vector as compared to wild-type virus to cells with or without the

appropriate receptor, in this case, a his-tag pseudo-receptor. This protocol demonstrates increased binding of modified virus to

receptor-positive cells (at levels greater than wild-type) with no loss of infectivity. Retargeted vectors can provide an additional tool

for increasing the efficiency of gene delivery to specific cell types.

� 2003 Published by Elsevier Inc.

Keywords: Gene therapy; Targeting; HSV; Virus entry; Glycoprotein; His-tag; Virion

1. Introduction

The advent of molecular biology has fostered the

development of a number of different methodologies to

transfer and express foreign DNA in mammalian cells.

Many early techniques were of limited use due to cel-

lular toxicity or the inability to transfect sufficientnumber of cells. Recent advances in technology have

reduced these limitations and many different cell types

can currently be transfected with high efficiency allowing

a broad range of basic scientific and therapeutic appli-

cations.

Perhaps, the most common technique for introducing

genes into mammalian cells relies on chemical means,

e.g., calcium chloride [1], and more recently, cationiclipoplexes [2]. These methods typically complex DNA to

a chemical entity, whether it be a salt precipitate or lipid-

* Corresponding author. Fax: 1-617-724-1537.

E-mail address: [email protected] (S. Wang).

1046-2023/$ - see front matter � 2003 Published by Elsevier Inc.

doi:10.1016/j.ymeth.2003.11.007

based moiety, and depends on endocytosis for cell entry

with subsequent escape from the endosomal compart-

ment. Similarly, DNA encapsulated in liposomes, which

also relies on endocytosis for cell entry, has been used to

deliver DNA into cells, and may be modified by incor-

porating viral envelope proteins to promote entry by

fusion [3]. Alternatively, generation of high electricalfields can facilitate uptake of foreign DNA into cells [4],

presumably by creating transient pores in the cellular

membrane for translocation to occur. Other sophisti-

cated techniques utilize physical means, e.g., with

biolistics, which bombards cells at high velocity with

DNA-coated gold particles [5], or by microinjection to

transfer DNA directly into the cell nucleus [6], for de-

livering genes directly into cells, but they tend to be costand/or labor restrictive. For most cell culture applica-

tions, any of these techniques will generally be useful for

experiments. However, even with these advanced tech-

niques, some types of cells in culture remain non-sus-

ceptible to gene transfer or the resulting transfection

efficiency remains too low or the process too toxic.

mail to: [email protected]

180 P. Grandi et al. / Methods 33 (2004) 179–186

Translating gene transfer experiments from cell cul-ture into animal models typically requires a higher level

of transfection proficiency. Efficient gene transfer in vivo

is more challenging since parameters such as cell mor-

phology, fluid dynamics, and tissue structure are very

different from those under standard cell culture condi-

tions. Consequently, many of the techniques that work

well in cell culture can be either toxic to tissues, limited

in distribution or be less efficient in transfection of thetarget cells when applied in animals.

Complementing non-viral delivery methods are a

number of recombinant viruses that have been modified

to deliver genes efficiently to many different cell types by

taking advantage of natural viral tropism. The first virus

commonly used to deliver genes into cells was retrovirus

(RV) [7]. Since then, adenovirus (Ad; recombinant and

‘‘gutless’’), adeno-associated virus (AAV), herpes sim-plex virus type-1 (HSV-1; recombinant and amplicon),

lentivirus (LV), and others have all been used to transfer

genes to different cell types with varying levels of success

[8–11]. In particular ‘‘gutless’’ Ad strain 5, AAV strain

2, HSV-1 amplicon, and LV vectors have shown the

most promise for gene delivery applications in the ner-

vous system [12]. Their advantages over non-viral

transfer methods include little-to-no toxicity and hightransduction efficiencies in vivo.

These vector-mediated gene delivery systems have

demonstrated widespread utility in many experimental

paradigms and some are available commercially (e.g.,

from Stratagene, QBiogene). One limitation of viral

vectors is that they require binding to specific cellular

receptors, dependent on the tropism of the virions, for

transduction to occur [13]. As a result, if the cells ofinterest lack the necessary receptor(s), then infection and

subsequent gene delivery is aborted. One technique to

overcome this potential limitation is to modify the nat-

ural tropism of the virus to recognize unique receptors

found on specific cell types [14]. This might allow in-

vestigators to achieve specific cellular expression of a

desired protein at high efficiencies.

For non-enveloped viruses, such as AAV and Ad, thatenter cells by receptor-mediated endocytosis, modifica-

tion of the capsid and/or fiber proteins can redirect vec-

tors to specific cell types [15,16]. Many of these

retargeting experiments have been developed and tested

usingAd vectors. For example,modificationsmade to the

Ad fiber protein to expose specific ligands have allowed

selective binding to cells expressing the complementary

receptor. These alterations can be extended to utilize abispecific antibody to recognize both a specific determi-

nant on the virion and a target receptor to achieve re-

targeting [17]. Exchanging viral serotypes can also be used

to retarget vectors. Recently, AAV-2-based vectors have

been successfully packaged with capsid proteins from

other AAV serotypes, such as AAV-4 and AAV-5, to

confer selective infection of different cell populations [18].

In contrast, enveloped viruses, such as HSV and RV/LV, typically fuse with the plasma membrane to deliver

capsids (and associated tegument proteins) into the cell

and can be retargeted to specific cell types by modifi-

cation of envelope proteins. The range of RV/LV cell

selectivity has typically been directed by exchanging the

viral envelope proteins with that of other viruses. A

common example has been to exchange the Moloney

leukemia virus envelope glycoproteins for the vesicularstomatitis virus envelope glycoproteins, which increases

virion stability and infectability [19]. Retargeting RV/

LV and HSV vectors is more challenging since any

modifications made to viral envelope glycoproteins must

be tested to confirm both binding to the target cell as

well as the ability to mediate fusion with the cell mem-

brane in order for gene delivery to occur.

HSV amplicon vectors have great utility for intro-ducing genes into cells and are relatively easy to produce

[20]. They have a large transgene capacity and can de-

liver up to 150 kb genetic information into a cell [21]. In

addition, no viral proteins are expressed from the am-

plicon as it contains only recognition sites necessary for

viral replication and packaging. HSV infection is initi-

ated by the binding of several glycoproteins, primarily

gC and gB, in the viral envelope to heparan sulfateglucosaminoglycan side chains on the surface of target

cells. Both gC and gB contain heparan sulfate binding

domains (HSBD), which are important for viral binding,

and when deleted decrease viral infectability [22].

Binding of the virus to the cell is followed by a mem-

brane fusion event between virion envelope and cell

plasma membrane, which is mediated by another gly-

coprotein (gD) in conjunction with gB, gH, and gL, anddelivers the de-enveloped capsid and associated tegu-

ment proteins into the host cell [23]. Efforts to retarget

HSV vectors have focused on deleting the HSBD in gC,

which is the primary determinant of binding, to mini-

mize native HSV tropism. By inserting a receptor-spe-

cific ligand into this domain, the modified vector should

bind to cells expressing the appropriate receptor and

allow selective transduction [24]. Other groups have alsotested HSV vector retargeting by deleting gB HSBD in

conjunction with ligand insertion into gC to further re-

duce natural tropism and achieve specific binding, but

these vectors had decreased infectability [25]. As more is

discovered about HSV biology and function perhaps

other glycoproteins could also be manipulated to re-

target to novel cell types, for instance, replacing gD with

vesicular stomatitis virus glycoprotein G (VSV-G)compromised infectivity [26]. Under optimal conditions,

native HSV tropism would be eliminated and only spe-

cific cell types would be infected at an increased inci-

dence relative to wild-type, unmodified virus.

Studies have shown that HSV can infect many cell

types both in culture and in vivo. It may be beneficial to

target a particular cell type both to increase efficiency

P. Grandi et al. / Methods 33 (2004) 179–186 181

and to potentially reduce the amount of virus in a givenapplication. For instance, under certain conditions, such

as tail vein delivery of virus, the vector is cleared rapidly

from the body with most taken up non-specifically by

liver cells [27]. If the viral envelope could be modified

such that the natural tropism is reduced and it binds to

target cells more efficiently, then more particles may be

able to infect the target tissues before being cleared by

the liver. Manipulating glycoprotein specificity usingcell-specific ligands has the potential to target infection

to many different types of cells. Here, a versatile method

to modify gC on any HSV-1 virion is described using a

his-tag ligand and a pseudo-his-tag receptor on cultured

cells as a model delivery system.

2. Methodology

Retargeting HSV amplicon vectors is a rapidly

evolving discipline. This protocol describes modifica-

tions made to HSV gC by peptide insertion–replacement

using a unique his-tag ligand to replace the HSBD. This

technology can be used to generate both retargeted

amplicon and retargeted recombinant helper virus vec-

tors. The basic scheme is illustrated in Fig. 1. In brief,cells are transfected with amplicon plasmid DNA con-

Fig. 1. Production of gC-modified virions. Amplicon plasmid DNA bearing

G418 selection for one week. The selected cells are then infected with replica

amplicon vectors, and are allowed to grow until 100% CPE is established. M

taining the gC gene with a unique ligand for a specificreceptor subtype fused in-frame with gC, replacing the

HSBD sequences, and selected to enrich for transfected

cells. Next, the cells are infected with an HSV re-

combinant helper virus that either does not express

functional gC or with one that does. During this step,

the helper virus provides in trans all the viral proteins

necessary for viral replication and packaging. The in-

troduction of the tegument protein, VP16, by the helpervirus infection activates the gC promoter and allows

expression and incorporation of modified gC into the

virion envelopes. The levels of modified gC produced by

the transfected amplicon DNA are much higher than

that for gC produced by the helper virus, such that even

with a gC-positive helper virus, most of the gC in the

resulting virions is modified. Retargeted virus can then

be harvested from the cultures and purified/concentratedfor experimental studies. This procedure results in a

mixed population of retargeted amplicon and helper

virus virions, both bearing the modified gC, in an ap-

proximate ratio of 1:100, respectively. Packaging meth-

ods are also available to eliminate the helper virus in the

preparation such that only the modified amplicon vec-

tors are produced [28]. Thus, this method is useful for

generating both retargeted amplicon or recombinantvirus vectors.

modified gC is transfected into vero cells, which are amplified under

tion-competent helper virus to produce gC-modified recombinant and

odified virions are then harvested and concentrated for analysis.


At this point, the vectors should be characterizedbefore use in experiments. Though not discussed here,

assays, such as Western analysis for proteins in purified

virions, should be performed to confirm incorporation

of the modified gC. And as with any new system, the

targeting must be tested empirically to determine if the

ligand is exposed for binding to its receptor on cells and

to confirm that infectivity of the vector via internaliza-

tion/fusion is not compromised.

2.1. Cell culture

Monkey Vero 2-2 cells (2-2), which express the es-

sential HSV-infected cell peptide (ICP) 27 necessary for

HSV-1 replication [29], are used to package modified

amplicon vectors and to propagate helper virus stocks.

However, any cell line, which is readily transfectablewith DNA, and permissive to HSV infection and repli-

cation can be used in these studies. Human 293 6H cells,

which bear a His-tag pseudo-receptor [30], are used to

demonstrate ‘‘proof of principle’’ for virion retargeting.

Both cell lines are maintained in 10% fetal bovine serum

(FBS; Sigma)/DMEM (Invitrogen) containing 0.5mg/ml

G418 (Invitrogen). Parental monkey Vero cells (ATCC)

and parental human 293 cells (Microbix) are maintainedsimilarly in the absence of G418. Penicillin (100U/ml;

Sigma) and streptomycin (100 lg/ml; Sigma) are added

to all media and cells were maintained at 37 �C in a

humidified incubator with a 95% air/5% CO2 mixture.

2.2. Recombinant helper viruses

The helper virus (gCD2-3) used in these experimentsbears a deletion in the gC gene with insertion of a CMV-

lacZ cassette to allow virus identification of infected cells

by X-gal histochemistry [31]. This virus is replication

competent, but is impaired in binding due to the lack of

gC when compared to wild-type. The absence of gC is

advantageous, as it allows incorporation of entirely

modified gC into the viral envelope without competition

from wild-type gC during amplicon packaging. Parallelgeneration of modified virions has also been carried out

Fig. 2. Cytopathic effect of helper virus. (A) 2-2 cells prior to infection. (B a

CPE for harvesting. Note that the two helper viruses have different phenot

gCD2-3 infection results in the formation of grape-like clusters (C).

with a virus bearing wild-type gC, hrR3, which alsobears a lacZ expression cassette [32]. In this method, any

recombinant HSV replication-competent helper virus

can be used to generate gC-modified amplicon and re-

combinant vectors, although careful assessment must be

taken with helper viruses expressing wild-type gC as to

the relative proportion of wild-type and modified gC in

the viral envelope.

Stocks of helper virus are prepared by plating 4� 106

2-2 cells in a 100-mm plate in the absence of G418 one

day prior to infection. On the following day, cells are

infected at a multiplicity of infection (M.O.I.)¼ 1

(1 transducing unit (tu)/cell) in 3ml for 4 h in normal

culture medium. An additional 6ml of normal culture

medium is then added to the inoculum after the infection

period. After approximately 16–24 h or when cytopathic

effect (CPE) is 100% (Fig. 2), the cells are scraped andmedia and lysate are collected. This mixture is then

frozen in a dry ice-ethanol bath and allowed to thaw in a

37 �C water bath to release virus from cells. Additional

virus is released from the mixture by sonicating the ly-

sate three times for 15 s in a bath sonicator (Fisher Sonic

Dismembranator 550, setting 3.5) filled with ice-slurry,

with a 1min cooling period in ice between sonications.

Large cellular debris is cleared by low speed 500g cen-trifugation for 10min. This total crude lysate is then

used as the starter inoculum to amplify virus from

1:6� 107 cells in 4� 100-mm plates at an M.O.I.¼ 1

and harvested, as described. The combined cell lysates

are then centrifuged through a 25% sucrose cushion (in

PBS) at 20,000 rpm (Beckman SW28 rotor; 72,000g)[33]. The resulting pellet is resuspended in 2ml PBS and

aliquots are stored at )80 �C. The virus is titered bycounting the number of lacZ+ cells as described below.

2.3. Titer assay

The 2-2 cells are plated as a monolayer in six-well

plates (5� 105 cells/well) in the absence of G418 the day

prior to assay. On the next day, cells are infected in a

total volume of 0.5ml normal culture medium withserial dilutions of virus, from 10�2 to 10�11 at 37 �C.

nd C) hrR3 and gCD2-3, respectively, infected 2-2 cells ready at 100%

ypes. hrR3 infection results in the formation of syncytia (B), whereas


After 2 h, the inoculum is removed and the cells arewashed twice with PBS and overlaid with normal

growth medium. Cells are fixed 24 h later in 4% para-

formaldehyde (Electron Microscopy Sciences) in PBS

for 10min at room temperature, washed twice with PBS

and stained with X-gal solution, pH 7.4, overnight at

37 �C [34]. The number of blue cells is counted to de-

termine titer (Fig. 3).

2.4. Amplicon plasmid

Modified amplicon plasmid, pCONGAH, was gen-

erated as described by Spear et al. [35] (Fig. 4). In brief,

an amplicon plasmid (pBON, kindly provided by Dr.

Dora Ho, Stanford University) [36] was modified to

contain a CMV-driven eGFP at a unique XhoI site. The

HSV-1 gC gene, including its own promoter, was sub-cloned upstream between PstI and HindIII sites in

pBON-eGFP. Next, the HSBD of gC was modified in

two steps. First, site-directed mutagenesis was per-

formed to insert a unique AscI site prior to gC codon 33

to create pCONGA. Then, the sequences encoding

amino acids 33-174 were deleted by AscI–EcoNI double

digestion to remove the HSBD coding sequences and

sequences encoding a six amino acid synthetic His-tag

Fig. 3. Amplicon and helper virus titering. The produced vector stock is seria

titers are assessed by counting eGFP+ cells at an appropriate dilution factor

after staining by X-gal. Two different magnifications are shown for each ser

increases. For example, using the 10� magnification, at 10�2 dilution for eGF

dilution. Bar is 0.2mm at 40� magnification and 0.5mm at 10� magnificat

containing a unique HpaI site were inserted to createpCONGAH (Fig. 4). The amplicon plasmid pCON-

GAH can easily be manipulated to insert novel ligands

into gC at the HpaI site to create a His-tagged fusion

protein or by replacing the His-tag by AscI–EcoNI di-

gestion and insertion of sequences for a peptide ligand.

Plasmid constructs are purified by QIAgen maxi col-

umns according to the manufacturer�s protocol and

checked by sequencing to confirm integrity and in-framecoding sequences.

2.5. Propagation of modified vectors

Amplicon plasmid encoding modified gC, for exam-

ple, pCONGAH, is transfected into vero cells

(4� 106 cells/100-mm plate) using Lipofectamine (Invit-

rogen). After one day, cells are cultured in the presenceof 0.3mg/ml G418 to amplify transfected cells. The

transfected cells are allowed to grow for one week under

drug selection. One day prior to infection, 1:6� 107 cells

(in 4� 100-mm plates) are cultured in the absence of

drug selection. On the following day, cells are infected at

an M.O.I.¼ 1 with gCD2-3 helper virus (or hrR3). Virus

is harvested when 100% cytopathic effect is evident and

concentrated as described above for helper viruses.

lly diluted and used to infect 2-2 cells as described. Modified amplicon

and modified helper virus is similarly assessed by counting lacZ+ cells

ial dilution to reflect the overall visual field area as the dilution factor

P+ cells, an equivalent number of lacZ+ cells are observed at the 10�4

ion.

Fig. 4. Diagram of amplicon plasmid pCONGAH. Sequences for

hexameric His-tag were inserted–replaced into the HSBD of HSV gC

(gC-His). This modified gC was then subcloned with its own promoter

into an amplicon plasmid bearing enhanced green fluorescent protein

(eGFP) and neomycin resistance (NeoR) expression cassettes [34].

Fig. 5. Cell surface binding capacity of CONGAH-modified re-

combinant virus. The binding capacity of CONGAH, hrR3, and

gCD2-3 viruses was evaluated on 293 and 293-6H cells at 4 �C over 10–

120min, followed by removal of virus by washing and recovery at

37 �C, as described in the text. Infected lacZ+ cells were identified by

X-gal staining [23].


Titers of resulting vector stocks, termed CONGAHv, aredetermined as described above, except that eGFP+

fluorescent cells are counted for amplicon vectors, and

lacZ+ cells are determined for helper virus (Fig. 3). This

protocol generates about 100 modified gC-containing

HSV virions, for every one modified amplicon vector. To

demonstrate proof of principle for virion retargeting, a

stable cell line expressing modified gC-His was produced

following transfection with pCONGAH by limiting di-lution under stringent G418 selection. A pure population

of lacZ+, gC-His modified virions is then generated by

infection with gCD2-3 virus, as described above, and

used to characterize binding and penetration.

2.6. Evaluation of modified vectors: binding and penetra-

tion assay

Monolayers of confluent 293 and 293 6H cells

(5� 105 cells/well in 6-well plates) are pre-incubated at

4 �C for 30min, washed twice with cold PBS, and then

incubated with the different virus stocks (CONGAH,

hrR3, and gCD2-3) at an M.O.I.¼ 0.1 in duplicate. The

viruses are allowed to bind from 30 to 120min at 4 �C,followed by three PBS washes to remove unbound virus.

The cultures are then shifted to 37 �C to allow for viruspenetration. Twenty-four hours later, lacZ+ cells were

counted to determine the number of cells infected.

3. Results and conclusions

Confirmation of HSV vector retargeting is provided

in Fig. 5, which shows increased binding of modifiedvector (CONGAH) specifically to cells expressing the

his-pseudo-receptor (293-6H) and more importantly,

enhancement in transduction efficiency that is greater

than wild-type gC virus. For every vector, transduction

increased over time the with highest binding and

transduction occurring after 120min for all treatment

groups without saturation. As expected, the gC-defi-

cient (gCD2-3) vector resulted in the lowest transduc-

tion rate for both parental 293 cells and 293-6H cells,although gCD2-3 is not completely impaired in trans-

duction, presumably due to other heparan sulfate

binding capacity of gB. This approximates previous

studies in which HSV-1 mutants lacking gC in the vi-

rion envelope binds with 60% reduced efficiency when

compared to wild-type virus [31,37]. Wild-type gC virus

(hrR3) infected both cell types approximately twofold

better than did gC-minus virus (gCD2-3) over the sametime points and represents natural transduction effi-

ciency. CONGAH vectors showed over a fourfold in-

crease in binding and infection of 293-6H cells when

compared to parental 293 cells, and to both cell lines

infected with either wild-type of gC-deficient viruses.

Thus, the addition of a His-tag in place of the HSBD

in gC was able to target entry of virions to cells ex-

pressing specific receptors and also increased infectivityover wild-type virus. These data were further confirmed

by His-tag competition assays as well as virus neu-

tralization assays [39].

These data suggest that gC-modified HSV virus vec-

tors can become a useful tool for delivering DNA into

specific mammalian cell types. The current packaging

system resulted in a 1:100 ratio of modified amplicon

particles to modified helper virus particles (Fig. 3)making it difficult to accurately assess amplicon retar-

geting per se by eGFP expression due to low amplicon

titers. Since the virion composition should be identical

for amplicon and recombinant vectors in the same stock,


modified gC binding efficiency was assessed using re-combinant vectors (via lacZ staining).

4. Future directions

This targeting technology demonstrates two useful

concepts: (1) the ability to increase the infectivity of HSV

for particular cell types beyond wild-type virus; and (2)the means to target infections to cells bearing specific cell

surface receptors. These capabilities are conferred by re-

placing of HSBD sequences in the gC envelope protein

with a peptide ligand. An amplicon has been created

which allows ready insertion of different peptide ligands

and generation of amplicon and/or recombinant virus

vector stocks expressing modified gC on the virion sur-

face. This methodology would be compatible with apeptide display approach to screen for novel peptides that

selectively increase infectability of a particular cell type in

culture or in vivo by propagation of amplicon and re-

combinant virus in specific cells or tissue, and subsequent

subcloning of enriched amplicons. It could also be utilized

to insert antigenic domains to use in combination with

antibodies that recognize that domain and determinants

on the cell surface. Furthermore, it should be possible toretrofit existing expression plasmids with non-coding

amplicon sequences necessary for amplicon replication

and packaging (i.e., oriS and pac), such that they can

subsequently be packaged and affect targeting to specific

cell populations.

This technology provides a two-edged sword. On the

one side, it can be used to target recombinant HSV

vectors. This could enhance the therapeutic potential ofoncolytic vectors by increasing delivery to tumor cells,

as they typically over-express specific receptors in their

cell membrane, such that systemically delivered vectors

would concentrate at the tumor site. On the other side,

when generating targeted amplicon vectors for delivery

to normal cells, the presence of helper virus could alter

cellular physiology and increase toxicity. In this case,

helper virus could be eliminated in two ways. First, oneor more therapeutic genes could be placed into the

pCONGA-derived amplicon expressing a modified gC

and packaged free of helper virus using the pac-minus

HSV BAC system [38]. This typically yields lower vector

titers, but these are adequate in many therapeutic

models. Second, one could take this therapeutic, gC-

modified amplicon and package it with helper virus

containing loxP-flanked pac sequences with final am-plification in cre-expressing lines [28]. This yields higher

titers of amplicon vectors with about 1% contamination

with replication-defective helper virus.

The upper size limit of ligand insertion into gC has not

yet been tested. It can be hypothesized that at up to 141

amino acids (the number of amino acids deleted from the

HSBD) can be inserted into the vector, although ligands

greater than 141 amino acids should be possible since theligands are exposed outside the viral envelope. In most

cases, each ligand subcloned into the vectorwill have to be

assessed individually as it is difficult to predict how fold-

ing of the new gC fusion protein will affect incorporation

into the vector, ligand binding, and vector retargeting. In

cases in which infective vectors are selected, e.g., using a

phage display approach, extensive testing may not be

necessary. HSV vectors have previously been retargetedby deleting gB HSBD and replacing gC HSBD with

erythropoietin (EPO) [25]. These vectorswere able to bind

to cells selectively, but were compromised in envelope

fusion and entered cells by endocytosis. Since both gCand

gB are vital to heparan sulfate (non-specific) binding to

cells, it might be useful to also modify gB, such that a

targeted ligand replaces the HSBD of gB and retains the

ability to fuse to cell and deliver the viral payload. How-ever, as mentioned previously, binding alone will not re-

sult in gene transfer, entry of the capsid and tegument

proteins would still have to be evaluated by assessing

downstream events, such as eGFP or lacZ expression.

The relative ease in constructing gC fusion proteins,

which replace HSBD with a specific ligand, in an ampli-

con plasmid with subsequent incorporation into virus

vectors should allow high-efficiency gene delivery to cellsonce unique receptor–ligand combinations are identified.

As it is, this system will allow easy testing of novel re-

ceptor–ligand interactions with the possibility of in-

creasing infection of the desired cell population. This can

be especially useful in enhancing oncolytic gene therapy

techniques or in transducing a particular cell type with

high efficiency. In addition to increasing infectivity of

HSV vectors for specific cell types, this method may alsoprove useful in purifying and concentrating virions by

incorporating a ligand to bind them to affinity columns.

Such purification epitopes could be combined with tar-

geting epitopes in the same virion.

This protocol does not eliminate replication-compe-

tent, infectious helper virus and should be carried out

under biosafety level 2 (BSL2) conditions, a level most

laboratories are comfortable with and have permissionfor. In contrast, helper-virus free modified amplicon

vectors [37] would be safe to use in the laboratory under

BSL1 conditions. This protocol represents a first step in

retargetingHSVvectors to deliver genes into specific cells.

In summary, a method is presented that can alter the

natural tropism of HSV vectors and permit infection of

targeted cell types beyond that of wild-type HSV vi-

ruses, and which can also be coupled to novel methodsof purifying vector stocks.

Acknowledgments

The authors thank Drs. David T. Curiel and Vickor

Krasnykh for providing the 293-6H cells and for


discussions related to theHis-pseudo-receptor. This workwas funded by NCI Grants CA86355 and CA69246.

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targeting hsv amplicon vectors

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