Note: Descriptions are shown in the official language in which they were submitted.
WO 95113391 , PCT/US94/12996
21 76284
I
Dr~r.~tit-n
METHOD OF USING, PROCESS OF PREPARING, AND COMPOSlTlON
COMPRISING A RECOMBINANT HSV-I
T ~ . h n i A I F i .~
The subject inYention is generally directed to a ~ HSV-I
suitable for use in nonmitotic cells and associated methods of treatment and processes for
preparation thereof. In particular, the subject invention provides l~ HSV-I
10 with a h-igh rate of expression of foreign gene sequences and/or a low cy~ JaiLi.,i~y in
neuronal cells.
nrollnfl of the Iny~ntif~n
The capacity to introduce gene sequences into a III^ I~ ;A I cell and to
15 enable the expression of the gene is of substAntial value in the fields of medical and
biological research. This capacity allows a means for studying gene regulation, for
defining the molecular basis for disease, and for designing a therapeutic basis for the
treatment of disease.
The illuudu~,Liull of a gene sequence into a ,~ host cell is
20 facilitated by first introducing the gene sequence mto a suitable vector. Vectors
suitable for use in nonmitotic cells, such as neural or neuronal cells, has proven
~AhA~ n~jnr Whereas most tissues in the body are readily accessible via the circulatory
system. the brain is shielded by the blood-brain barrier and peripheral nerve cells may
be encased in a myelin sheath. These physiological barriers, along with the non-
25 replicative state of most nerve cells, present peculiar challenges when designing genetherapy systems.
These challenges have hindered the possible treatment of ll~ulo~
disorders such as brain tumors, d~ aLiv~ disorders (multiple sclerosis, Parkinson's
disorder, Al~heimer's disorder (Tanziet al., Sci., 235:880, 198?), a~ LIu~JlliC lateral
30 sclerosis)), disorders caused by abnormal expression of genes, inherited disorders
caused by a known gene defect, (HPRT in Lesch-Nyhan disorder; I~ IA (Leeet
al., Sci 235:1394, 198?); ~lu~,o~ luulu:,idase (Sorge et al., Proc. NatL Acad. Sci. USA
84:906, 1987); and Duchenne's muscular dystrophy (Monacoet al., Nafure 321:443,
1986)) and acute injuries to the brain or peripheral nervous tissue, for example from a
35 stroke, brain injury, or spinal cord injury, all of which may be treatable using gene
transfer techniques.
S~STITUTE SHEEr f~RUI 2f~
WO 95/13391 2 ~ 7 6 2 ~ ~ PCTIU594/12996
~ '. . ., ~
Althcugh many viral vector systems have been developed, there has
been difficulty adap-mg these systems to use in neuronal cells. Retroviral vectors have
been used to tr~msfer genes into neuronal cells in vifro (Priceet al., Proc. NatL Acad Sci
USA, 34: 156-160, 987), and in vivo (Culveret al., Science, 2~6:1550, 1992); Priceet
5 al., supra), they have not proven useful in delivering genes to a large proportion of cells
in the nervous system. Other viral vector systems also have . I IA A~ limiting
their usefulness for gene transfer into neuronal cells, such as: rapidly clearing Iytic
infections (e.g., adenovirus, vaccmia virus), small genome size (SV40, polyoma), or
limited cell tropism (EBV, bovine papilloma virus).
A Herpes Simplex Virus-l (HSV-I) vector has been shown to be useful
for infecting a wide variety of cells, including neuronal cells (Spear and Roizman, DNA
Tumor Viruses, Cold Spring Harbor Laboratory, NY, pp. 615-746). HSV-I can exist in
a latent state in neural cells (Stevens, Microbiol. Rev. 53: 318~ 1989) allowing for stable
e of the vector. Additionally, the viral genome of HSV-I is ver~ large (150
kb) and may ~ mml~' large nucleic acid segments. Plasmid-based HSV-I vectors
have been construc-~ed, but have several major drawbacks. In particular, they caîmot
easily establish latency, reducing the chcnce of long-term expression in target cells.
Moreover, they require a helper virus for packaging which carlnot be totally eliminated
from the plc~ lLivll. In addition, helper viruses may exert cytopathic effects on the
target cells.
Geller et al. (PCT WO/90/09441) developed a HSV-I virus-based
vector, which, while offering advantages over plasmid-based vectors, has failed to be
efficacious in several instances. These vectors suffer from promoter i~ ,y and
high uyLvlJ~Lll;~iLy~ -hus severely limiting their use m gene transfer. While others have
tried to increase ex~ression by using a variety of p}omoters (Tackney, et al, J: Virol.,
52: 606, 1984), cy;~ ,;Ly has been shown to be a persistent problem, even in those
viral vectors which are replication deficient (Johnsonet al., J. Virol. 66: 2952, 1992,
Johnson et al., Mol Brain Res., 12: 95, 1992). For long-term expression in neuronal
cells, it is necessary to have a viral vector that ' low ~ op_Lll;~.;Ly
In view of the inability of current HSV-I vectors to adequately account for
the balance of uyLu~lLllh,iLy and gene expression, it is apparent that there exists a need for
newandadditionalmethodsandA."..~ ;;,.,..cwhichaddressandrectifytheproblem.
The present invention fulfills this need, and further provides related advantages.
SIJBSI 11 llTE SHEET fflULE 26)
WO 95113391 PCI/US94/12996
~ 2 1 76284
S.-mmA~ y of the Tnventi~An
The present invention is directed to ~ Herpes Simplex Virus-
1 ("HSV-I") capable of drrecting expression of a G protein linked receptor gene.Within certain l~ o~ of the invention, the ,,.~...,1.;.,--,~ viruses direct the
5 expression of such genes in non-mitotic IIIAI.IIIIA~;A" cells, and more preferably, in
IIIAI~IIIIAI; - ~ neuronal cells.
Within other aspects of the present invention, lc~ HSV-I are
provided which are capable of directmg the expression of an antisense transcript of the
G protein linked receptor gene.
In one rl.ll.o,l;,.. l of the invention, l~ HSV-I are provided
which are deficient for the expression in one or more of the following: thymidine
kinase; virion host shut-off protein (VHS); or a replication loci. such as that for ICP4
protein
In another emhoriimPnt of the present invention, the gene encoding a G
protein linked receptor or a antisense segment thereof is inserted in the TK locus of the
HSV-I vira~ genome. For example, the antisense segment may be a 5-HT2 receptor
gene. Numerous G-protein linked receptor genes may be utili~ed within the context of
the present invention, including, by way of example, a human Ml muscarinic
acetylcholine receptor gene or an adrenergic receptor.
Within other aspects of the invention, methods of treating mammals for
neurological disorders are provided, comprising the step of a,l...;,.: . ~ . ;"g to a mammal
a c.,."~ ;l;.)" comprising a ~ HSV-I, within certain rl"l-~,l;l,l..l,~, in
with a ~ Ally acceptable carrier or diluent~
Within certain ~ t~.l; .l l. .1.~, the a.l. ll;..;~l. Al ;.,.~ of l~
...... ~ :l;.,.. cmaybeA~ iby,forexample,by~ t"- l;.Ailyllli.,lu;llJc~,Lvl.,
a trme release 11~ II, a sustamed release rA~PAhAAnicm chronic infusion, or ex vivo
I l IA . I ~ I ~ IAI; -I ~ cells infected with a ".. 1,;, ~ I HSV-I .
Another aspect of the present invention provides l .l "..,, ~A. . . ~ ;~ AI
C-~ UIII~ a ~ 1 vrrus of the present invention and a
l.1~ IY acceptable carrier or diluent.
Within yet other aspects of the present invention, processes of producing
HSV-1 with low tyLu~L1.L;~,;Ly are provided, comprising the steps of
culturing ,,.A,.. ,AI;A.. cells with a first rt~ .. l.;. - .l HSV-I virus containing a G protein
linked receptor gene and a second ~ HSV-I virus defective in a gene
35 required for replication under conditions and for a time sufficient to allow
.,...I.:..A.;on of the first and second viruses; and, selectmg the l~ virus by
S~STl~UTE SHE~T (RULE 26~
WO 95/13391 PCTlUSg4/12996
` 2 ~ 76284
detecting G protein inl~ed receptor expression. Further, the G protein linked receptor
gene can be inserted nto the TK locus. Within certain r~ fU~ , the first virus may
be vhsA and the second virus may be dl20.
Another aspect of the present invention is a process wherein the first
5 lC 1 virus is deficient in the expression of one or more of the following: theTK locus, the virion host shut-off protein ~VHS), and the replication loci, such as that
for ICP4 protein.
Other aspects of the present invention provide l' "..I ,;",..,l HSV-I with
an in ~itro ~;y~uuailfi-,;Ly generally less than about 3%; typically in the range of 0.1% to
1.0%; and preferably in the range of about 0.001% to 0.1%.
Withi~ yet other aspects, l~ HSV-I are provided which are
capable of expressing a G protein linked receptor with a surface receptor expression
generally of greater than 10,000 ~ ul~/ccll; typically in the range of 25,000-200,000
,U~Ul~/C~ II; and preferably in the range of 200,000 to 400,000 I~ Lul~
Yet other aspects of the present invention provide methods of using
.;11, 111 HSV-I in the ,-,--"-~ of a ",~ ~I f -' 1- "I for the treatment of neuronal
disorders.
These and other aspects of the present invention will become evident
upon reference to the following detailed description and attached drawings. In addition,
various references are set forth which describe in more detail certain procedures and/or
r.~ and are hereby ;.l~ull ' by reference in their entirety as if each were
specifically ~ by reference.
I~r~crrirfirm of F~~ s
Figure la is a schematic illustration of vhsA.
Figure Ib is a schematic illustration of vTKhml-l .
Figure Ic is a schematic illustration of vTKhml-2.
Figure Id is a schematic illustration of vTKhml-3.
Figure 2 is a schematic diagr~m illustfating the detection of mlACHR 5'
30 mRNA using a . ;1,~ protection assay.
Figure 3 is a rh~tQ~ :~rh of a urea/polya~,.yl,lllfid~, gel which shows
labeled probe that had hybridized to cellular RNA was ~ y identified
following cl_~,LIu~l~ul~;, on an 8M urea/pol~a.,.yla",;de gel and visualized by
fl~1;r~ y A p}otected RNA fragment of 265 nt which Cull~f.~JUlld~d to
35 ~ , of the insert from the CMV promoter was detected as early as 3 hours post
S~lBSTllUTE SHEEr (RU E 263
WO 9~/13391 PCI~/US94/12996
2 1 76284
infection ("hpi"), reached high levels by 8 hpi, and maintained high levels until 18 hpi.
(See Example 2.)
Figure 4 is a graph which shows saturation curves lcp~ g the
number of mlAchR expressed per Vero cell in samples harvested 2 to 36 hours post-
5 infection (hpi) in samples infected with one of the following: vTKhml -I, vTKhml-2,
and vTKhml-3.
Figure S is a graph which shows saturation curves lcplc~ l the
number of mlAchR expressed in t;ansfected E5 cells in samples harvested 2 to 20
hours post-infection (hpi) in samples infected with one of the following: vTKhml-l,
10 vTKhml-2, and vTKhml-3.
Figure 6 is a bar graph which shows the number of mlAchR expressed
in primary cortical neuron cultures at 12 hpi for vhsA, vTKhml-l, vTKhml-2, and
uninfected Vero cells.
Figure 7 is a graph which shows saturation curves lC,ul.~Cllliug a
15 ~ of receptor binding of vhsA to vTKhml-l .
Figure 8 is a lullu~ut~a~ of a a southern blot of viral DNA, comparing
vhsA and vTKhml-l.
Figure 9 is a 1~ IAIIII which shows a field of primary mouse cortical
neurons growing on glass coverslips infected with vTKhml-3. Briefly, cells growing
20 on glass coverslips were rinsed with isotonic salme and fixed with 3.2% formaldehyde
for 10 mm at room ~~ L~.. Cells were rinsed and ~ ;" ~I with 0.3% Triton
X-100 for 3 min at room t~,lllu~ . Cells were then rinsed and incubated in primary
amtibody for I h, rinsed three times with saline, and incubated with fluorescentantibodies for I h at room t` ~''I'' '-l'''c Following this incubation, cells were rinsed,
25 mommted on a glass slide amd viewed using an ~;nuul~ e uu~,lu~-,U~ vith barrier
filters to distinguish green from red nuul~.,.ll~,c. The green signal is derived from
fluorescein-;,ullllo."~ conjugated goat anti-rabbit amtibody non-covalently attached
to the primary rabbit polyclonal antiserum anti-enolase. The oramge signal is derived
from tetramethyl rhodamine i~u~uO~"y conjugated goat anti-mouse antibody
30 attached non-covalently to a mouse ~ IIAI antibody directed against the herpes
protein ICPO.
Figure 10 is a Illl..l~.~lAllll of a gel which shows protein synthesis in
infected cells ~ ;llg that vTKhml-2, which is the backbone vector for
vTKhml-3 and vTKhml-l, does not alter protein synthesis after infection. Monolayers
35 of Vero cells were infected with virus for I h at 38, and rmsed with growth medium.
Cells were then incubated with growth medium lacking cold m.-thi-~nin~ After 30 min,
SUBSrlTUTE SltEET (RULE 26~
W095/13391 ~ r ~ PCT/US94/12996
2 ~ 762~4
100 mCi/ml [35S] " ~ was added for the remainder of the e~rP~imPnt Cells
~vllol~l~ were harvested in detergent buffers and proteins were identified on SDS
gels.
Figure 11 is a photograph of a DNA replication assay confirming the
phenotype of each strain of virus. Briefly, the results of this assay show that vTKhrnl-2
and vTKhml-3 do not replicate in normal Vero cells, but do replicate in E5 cells, which
express ICP4 and ~ the defect in the virus.
~)Pt~ilP~i Deqrriq~tirn oftheTnvPnfinn
wit 1in the various aspects of the present invention, Ir~ Herpes
Simplex Virus-l ~SV-I), is utilized as a means of illLIudU,,ill~ nucleic acid segments
into nonmitotic cells primarily of the nervous system (collectively referred to as
"neural" or "neuror~al" cells). Specifically, ~ ..,..l.;",.,.l HSV-I ofthe present invention
acts to deliver nucleic acid segments into the cell where the proteins are expressed,
generally as mRNA which is then translated into a protein. When the protein translated
is a G protein linked receptor, for example, fhe protein enters the secretory pathway of
the host cell and is expressed on the cell surface as a receptor. The receptors are in the
correct orientation to bind their associated ligand and linked to a second messenger
system and, thus, function in much the same manner as a naturally occurring receptor.
Briefly, HSV-I is a double stranded DNA virus (approx. 152 kb) which
is replicated and transcribed in the nucleus of the cell. The HSV-I genome is described
in detail in Fields et al., ~ . ~ T Virology, Raven Press, N.Y. (1986). The specific
strain of HSV-I er~ployed as a starting material in the present invention is not critical.
One suitable exam~le is the KOS strain.
Productive infection by HSV-I usually results in cell Iysis or alteration
of host l~l~lul~lalr..,~ processes. However, HSV-I also may be n~ inPd
i ldc;L~.~,ly in the 'latent state" in certain cells by a mPrh~niqm involving the tegument
of the virus pa~ticles. Tbe .~Li~ iu.. of the virus is regulated by certain systemic or
cellular events. The latent virus is still l~ AIIY active, producing "latency
30 associated transcri~ts" (LATS). Mutant viruses that are ~;ul~llulu~ll;,,~,J or defective in
their replication potential can still enter the latent state (e.g, UL41(-), TK(-), and
ICP4(-)). In fact, a TK(-) HSV-I will maintain the latent state illv~:rll~.~.ly. Thus,
HSV-I is ideal for use in delivering nucleic acid segments to non-mitotic cells such as
neuronal cells. ~'ithin the present invention, HSV-I is preferably maintained in the
35 latent state.
SUBSTITUTE SHEET (RULE 26)
wo gSrl3391 PC'r/US94/12996
~ 2t76284
The ~ nn of HSV-I for the purposes of the present invention,
primarily involves the ~ regions of the HSV-I genome, generally
IIIA;~.~-;~I;II~ the essential regions intact. In the context of the present invention,
"essential region" refers to any region of the viral genome tne deletion of wbich would
S result in an inability to infect a ~ host cell or an inability to replicate, even
with the assistance of a helper virus or a ~ cell line. Nv~ clliial regions
within the genome may, but need not be, deleted in whole or in part.
Within the context of the present invention, the term "helper viruses"
refers to replication competent infectious viruses that provide gene products required
10 for the plu~ 5aLiUII of replication defective viruses that can not, by definition, propagatc
themselves. Such helper viruses are described in Fields et al., Fu, ~ t Vlrology,
Raven Press, N.Y. (1986) and are well known to tbose skilled in the art. Examples of
helper viruses suitable for use in tbe present invention include unaltered HSV-I as well
as other viruses that express the genes contained witbin the deleted region whose
15 products are necessary for ulU~ua~aiioll of l~ HSV- I .
The term ".. l.! .. l;.. A~ cell lines" refers to cell lines that provide
gene products required for tbe lulupa~ iull of defective viruses that by definition cannot
propagate themselves. Suitable ....,.l,i.."..,l;"~ cell lines in the present invention
include E5 Vero cells, which provide the protein ICP4 for replication deficient viruses.
20 (Disclosed in detail in DeLuca et al., J. of T~iroL 56:558-570 (1985)).
As noted above, within certain aspects of the present invention, nucleic
acid segments are inserted into tne HSV-I genome and/or portions of tne HSV-I
genome are deleted. Preferably, insertions or deletions of nucleic acid segmentsutilized in the present invention are made to one or more of tbe following ~
25 regions: the UL41, thymidine kinase (TK), amd/or any one of several replication loci.
IAhe replication loci mclude DNA polymerase and that for the ICP4 protein. Briefly,
ICP4 is a protein produced by an ' -~Iy gene and governs l~
regulators required for the expression of the early genes. Thymidine kinase is an early
gene implicated in the replication of viral DNA. UL41 is a late gene whose protein
30 product is l~ pUI~ibl~ for early shut off of host cell ~ ,lulllùl.,.,lllal synthesis.
The HSV-I genome can be r .' ' to produce such deletions and
insertions by using standard .~ l DNA techniques, such as those described in
Maniatis et al., Molecular Cloning, ,4 Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1982). Briefly, deletions witbin HSV-I genome
35 cam be effected by ;ul~ iull_l techniques employing ~ ~..1...,.~. l. . ~. ~ ~,.., .. l.... ~ amd
the like. Insertions can also be executed using cullv~,..L;ullal techniques, including, by
SUFSTlTUTE SltEEr (RULE 26)
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'"I`S 2176284
way of example .,ul ,,., r~ l ;", . i.e,, 1,.., ..nlh~,,....~ 1,=~ ..., .1.:., -: ;. ,, . facilitated by a suitable
plasmid. A suitab e plasmid available for such use includes pRc/CMV (Invitrogen
Corp.). The plasmid including the desired ~ r~ cam be selected using
~,u~ iullal methods and introduced for ~lu~a~ iull purposes into a host cell or
S organism using standard l,,.,.~r(.,...Al;nn procedures. The plasmid is then isolated from
the host organism, mixed with unaltered HSV-I DNA and ~ 1 into host cells.
The cells containin- the plasmid and the HSV-I DNA are cultured, and l~...l.nln~;,.,.~
take place, resulting in the l~pla~,~,.ll.,ll~ of the unaltered region in the
viral DNA with the CUIICaPUIId;ll~ altered region from the plasmid. Any host cell
10 suitable for plasmid and HSV-I DNA l.~ ... and subsequent l~ virus
UlU~ liiUII can be utilized in this procedure. The l,~ ..,.,l.;"A,.I HSV-I DNA is then
replicated within the cell and the viruses which have undergone the desired
.",,1"",.".." are selected using standard techniques.
As noted above, 1~ l HSV-I of the present invention are
15 produced through .nsertion of nucleic acid segments into the genome. Within the
context of the present invention, "nucleic acid segment" refers to a nucleic acid
molecule derived from a variety of sources including DNA, cDNA, synthetic DNA,
RNA, or cnmhinsn nnC thereof. Such nucleic acid segments may comprise genomic
DNA which may or may not include naturally occurring introns. Such genomic DNA
20 may be obtained in association with promoter regions or poly A sequences. Further,
The nucleic acid segment may be an antisense sequence. The nucleic acid segments of
the present invention are preferably cDNA. Genomic DNA or cDNA may be obtained
in any of several ways. Genomic DNA can be extracted and purified from suitable cells
by amy one of several means. Alternatively, mRNA can be isolated from a cell and used
25 to produce cDNA 'cy reverse ~ r by any one of several methods.
Within particular preferred ,,,.l..~.l;,,,..,l~ of the present invention, the
nucleic acid segment is a G protem linked receptor gene. In the context of the present
invention, the term "G protein linked receptor" refers to a guanine nucleotide binding
regulatory protein coupled to both a cell surface receptor amd an effector, such as an ion
30 channel, together comprising a IlAII~ lr signaling system. G protein linked
receptors mediate the actions of ~tr~rrll~ r signals, such as ~ ~ul~ ;lr~ l~ They
are described in ~etail in Dohlman etal., Ann. Rev. Bioci?e~n. 60:553-588 (1991).
Suitable G protein linked receptors genes include those listed in Table I and portions
thereof.
SU13SrlTllTE SHEEr (RULE 26
WO 95~13391 ; ~ , , . . PCT/US94/12996
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It will be evident to those skilled in the art tnat tne particular receptor
utilized will be influenced by the ~ of the receptor and the specific
treatment.
S TABLE I
Receptor Subtype Species Ret'.
~amma~
~31-adrenergic Humam Frielle, T., et al., Proc. Natl Acad SCL USA
84:7920-24, 1987.
Rat Machida, C.A., et al.. J B~ol Chem. 2~5:12960-
65, 1990.
~32-adrenergic Hamster Dixon, R.A.F., et al., Na~Yre 32~:75-79, 1986.
Hmnan Kobilka, B.K., et al., Proc. NatL Acad SCL USA
84:46-50, 1987.
Schofield, P.R., et al., Nucleic Acids Res. 15:3636,
1987.
Churlg,F.Z.,etal.,~EBSLett. 211:200-6, 1987.
Emorine, L.J., et al., Proc. NatL Acad Sci USA
84: 6995-99, 1987.
Mouse Allen,J.M.,etal.,EMBOl 7:133-38,1988.
Rat Gocayne, J., et al., Proc. NatL acad Sc~ USA
84:8296-300, 1987.
Bucklarld, P.R., et al., Nucleic Acid~ Res. 18:682,
1990.
133-adrenergic Human Emorme, LJ.. et al..... Science 245:1118-21~ 1989.
Ig-adrenergic H~unster Cotecchia, S., et al., Proc. NatL Acad &L USA
85:7159:63, 1988.
Rat Voigt, M.M." et al., NYcleic Aci~s Res. ~8:1053,
1990.
Ic-adrenergic Cow Schwimm,D.A.,etal.,J.BioLChem.265:8183-89,
1990.
2A-adrenergic Hmmam Kobilka, B.K., et al., Science 238:650-56, 1987.
Fraser, C.M. et al., J. BioL Chem. 264:11754-61,
1989.
Rat Chalberg, S.C.,et al., MoL CelL Biochem. 97:161-
72, 1990.
Pig Guyer, C.A., et al., 1 BioL Chem., 265:17307-17,
1990.
2B-adrenergic Humam Regan, J.W. et al., Proc. Nat'L Acad Sci USA 85:6301-5, 1988.
Rat Zeng, D.W.et al., Proc. Na~'l Acad Scl. USA
8~:3102-6, 1990.
2C-adrenergic Humam Lomasney, J.W.et al., Proc. Nat'l. Acad &L USA
87:5094-98, 1990.
5-HTI a ~ Humam Kobilka, B.K., et al., NatYre 329:75-79, 1987.
Fargin, A., et al., Nature 335:358-60, 1988.
Rat Albert, P.R, et al., 1 BioL Chem. 265:5825-32,
1990.
SUBSTITUTE SltEET (FWLE 26~
WO 95/13391 2 1 7 6 2 ~ 4 PCT/US94112996
5-HTlc-serotoner~ic Rat Julius, D., et al., Science 241:558-64, 1988.
5-HT2-serotonergic Rat Pritchett D.B,etal.,EMBOJ. 7:4135-40,1988.
Julius. D. et al.. Proc. Natq Acad SCL USA
87:928-32, 1990.
Ml-muscarinic Pig Kubo,T.,etal.,Na~Yre323:411-16, 1986.
Human Peralt~7 E.G7 et al., EMBOl 63923-29,1987.
Allard, W.J., et al., Nuclelc Aculs A7es. 15:10604,
1987.
Rat Bonner, T.l., et al., Science 23 7:527-32, 1987.
Mouse Shapiro, RA., et al.. J. BloL Chem. 263:18397-
403, 1988.
M2-muscarinic Pi6 Kubo, T., et al., FEBSLet~. 209:367-7Z, 1986.
Peralta, E.G., et al.~ Science 236:600-5, 1987.
Human Peralt~7 E.G., et al., EMBO J 6:39Z3-Z9, 1987.
Rat Gocayne, J., et al., Proc. Nat'L Acad. Sci USA
84:8Z96-300, 1987.
Bomner, T.l., et al., Science 237:527-32, 1987.
M3-muscarinic Human Peralta,E.G.,etal.,EMBOJ.6:39Z3-29,1987.
Rat Bonner, T.l., et al., Science 237:527-32, 1987.
M~ Human Peralta, E.G., et al., EMBOl 6:3923-29, 1987.
Rat Braun, T., et al., Biochem. Biophys. Pes. Commun.
149:125-327 1987.
Pig Akiba, 1., et al., FEBS Left. 235:257-61, 1988.
M5-muscarmic Humim Bommer, T.l., Neuron. 1:403-10, 1988.
Rat Bommer, T.l., Neuron. /:403-10, 1988.
Liao, C.F., et al., 1 BioL Chem. 264:73Z8-37,
1989.
Dl .', ,, Human Dearry,A.,etal.,Nature347:7Z-75, 1990.
Zhou, Q.Y., et al., Nature 347:76-80, 1990.
Rat Zhou,Q.Y.,etal.,N~ture347:76-80, 1990.
O'Dowd, B.F., etal., FEBSLett. 347:8-lZ, 1990.
Dz-', ~- Rat O'Dowd,B.F.,etal.,~EBSLett.347:8-lZ,1990.
Todd, R.D., et al., Proc Nat'L Acad Scr USA
86:10134-38, 1989.
Human Todd, RD., et al., Proc. Natq Acad. ScL USA
- 86:10134-38, 1989.
Grandy, D.K., et al., Proc. Nat'L Acad Sci USA
86:9762-66, 1989.
alterna- Monsma7 F.J., Jr., et al., Nature 342:9Z6-29, 1989.
tively Miller, J.C., Biochem. Biophys. Res. Commun.
spliced 166:109-lZ, 1990.
D3- ~ Rat Sokolof~, P., et al., Nature 347:146-51, 1990.
Substance K Cow Masu, Y., et al., Nature 329:836-38, 1987.
Rat Sasai, Y., et al., Biochem. Biophys. ~es. Commun.
165:695-702, 1989.
Human Gerard, N.P., et al., J BioL Chem. 265:Z0455-62,
1990.
Neuromedrn K Rat Shi~emoto, R, et al., 1 BioL Chem. 265:623-28,
1990.
SubstanceP Rat Yokota,Y.,etal.,J. BioL Chem. 264:17649-5Z,
1989.
Hershey, A.D., et al., Science 247:958-6Z, 1990.
F-Met-Leu-Phe Human Thomas, K.M., et al., 1 BioL Chem. 265:20061-
64, 1990.
Sll~SrllUTE SHEET (RULE 26~
WO 95113391 PCTIUS94/12996
.. ~ ; 21 762~4
11
Thyrotroprn Dog Parmentier, M., et al., Science 246:1620-22, 1989.
Libert, F., et al., MoL Cell. Endocrmol 68:R15-
17, 1990.
Hu!nan Libert, F., et al., Biochem. Biopf~s Res.
CommYnl6S:1250-55 1989.
Nagayama, Y., et al., Biochenm. Biopl~s. Res.
Commun. 165:11845-90.
Rat Akaunr~u, T., et al., Proc Natq Acad. Sci USA
87:5677-81, 1990.
Lutroprn~ ,, Rat McFarlarld, K.C., et al., Science 245:494-99,
1989.
Pig Loosfelt, H., et al., Science 245:525-28, 1989.
Endothelrn Human Mrnegiah, T., et al., Biochem. Biopiys. Res.
Commw. 172:1049-54, 1990.
Cow Arai, H., et al., Naf we 348:730-32, 1990.
Endothelin-ETg Rat Sakurai, T., et al., Na~ure 348:732-35. 1990.Angiotensin (m~s) Hunlan Young, D., et al., Cell 45:711-19, 1986.
Jackson, T.R., et al., Na~ure 335:437-40, 1988.
Rat Young, D., et al., Proc. Na~'l. Acad Scl USA
85:5339-42, 1988.
Rhodopsrn Cow Hargrave, P A., Pro& Re~inal Res. 1:1-51, 1982.
O~chinnikov, Y.A., FEBSLe~t 148:179-91,1982.
Nathans,3.,etal.,Cell34:807-14, 1983.
Hurnan Nathans, 1., et al., Proc. Na~'l Ac~d. Scl USA
al:4851-55, 1984.
Mouse Bæhr, W., et al., rEBSLe~. 238:253-56, 1988.
Redopsin Human Nathans,J.,etal.,Science232:193-202,1986.
Green opsrn Human Nathans, J., et al., Science 232:193-202, 1986.
Blueopsrn Hunlan Nathans,J.,etal.,Science232:193-202, 1986.
Cannabrnoid Rat Matsuda, L.A., et al., Na~ure 346:561-64, 1990.
Unknown-RDC I Dog Libert, F., et al., Science 244:569-72, 1991.
Urlknown-RDC4 Do~ Liber~,F.,etal., Science244:569-72 1991.
Unkr~own-RDC7 Dog Liberl, F., et al., Science 244:569-72, 1991.
Unknown-RDC8 Dog Libert,F.,etal., Science244:569-72 1991.
I l ' .. ~l~l Human Hla, T., et al., J. Bio. Chem. 265:9308-13, 1990.
Unknown-RTA Rat Ross, P.C., et al., Proc Na~'l. Acad. SCL USA
87:3052-56 1990.
. ..
Adrenergic (~1-) Turkey Yarden, Y., et al., Proc. Na~'L Acad. SCL USA
83:6795-99, 1986.
Serotonergic Fly Witz, P., et al., Proc. Na~'L Acad. Sci USA
87:8940-44, 1990.
Muscarinic Chicken Tietje, K.M., et al., J. BioL Chem. 2 2828-34,
1990.
Fly Shapiro, R.A., et al., Proc Na~'L Acad SCL USA
86:9039.
Onai,T.,etal.,FEBSLe~.255:219-25, 1989.
Opsrn (ninar~) Fly O'Tousa, J.E., et al., Cell 40:839-50, 1985.
Zuker, C.S., Cell 40:851-58, 1985.
Opsin-Rh2 Fly Cow!nan, A.F., Cell 44:705-10, 1986.
Opsrn-Rh3 Fly Zuker, C.S., et al., Neurosci. 7:1550-57, 1987.
Opsrn-Rh4 Fly Fryxell, K.J., et al., EMBO J 6:443-51, 198_.
Montell,C.,etal.,~Neurosci. 7:1558 .
SJBSTITUTE SHEEr (RULE 26'~
WO 9~/13391 t ~ . ~ 21 7 6 2 8 4 PCTIUS94112996
i~hodopsrn Fly Ovchinniko~r, Yu.A., et al., FEBS Leff. 232:69-72, 1988.
Chicicen Tai~ao, M., et al., Vislon Res. 28:471-80, 1988.
Octopamrne Fly Arakawa, s., et al., Neuron 4:343-54, 1990.
Mating factor (ST--2) Yeast Marsh, L., et al.~ Proc. Nat'L Acad SCL USA
87:3855-59, 1988.
surichoider, A.C., et al., Nucleic Aclds Res.
13:8463-75, 1985.
Nai~ayama N ., et al., EMBO J. 4:Z643-48, 1985 .
(STE3) Yeast Nai~ayarna, N ., et al., EMBO J. 4:2643-48, 1985.
ilagen, D.C., et al., Proc. Nat'L Acad. Sci. USA
83:1418-22, 1986.
cAMP Slr ne mold Klein, P.S., et al., SCfence 241:146-72, 1988.
Uni~nown-US27 Viral Chee, M.S., etal.,Nature344:774-77, 1990.
Uni~nown-US28 Viral Chee, M.S., et al., Nature 344:774-77, 1990.
Uni~nown-UL33 Yr~al Chee, M.S., et al., Nature ~44:774-77, 1990.
Although it is pre*rable to utilize the complete coding sequence from
the G protein linked receptor gene, within certain r,l,l)oll;ll. ~ of the invention only
S that portion of the G protein linked receptor gene which encodes expression of the
receptor on the cell surface need be utilized. Within the context of the presentinvention, both the entire coding region and portions thereof are referred to as "G
protein linked rece~tor genes." Such expression can be determined by any one of
severai suitable meæns, includmg ligand binding assays.
The coding sequence for the G protein linked receptor should be inserted
in such a manner tlat the resulting lC ~,,,1.;.,--: HSV-I genome contains a promoter
upstream from the coding region of the G protein linked receptor sequence and the
coding region of t~le G protein linked receptor sequence in the reading frame. The
desired G protein Iinked receptor produced should be compatible with HSV-I
15 IJlU~l..~,..~iUII (i.e., i3 not lethal). The promoter sequence can be supplied within a
separate or the sam nucleic acid segment a~3 the G protein linked receptor sequence or
by the HSV-I genomic portion of the l.,~ virus. Suitable promoters include
any one of severai which are capable of initiating expression of the G protein receptor
gene. Preferably, tle promoter is a major irnmediate early promoter and the sequence
~0 includes a pOl~a i.,ll~laliu.l site. More preferably, the promoter is the cylulll~,~ivv;lu:~
(CMV) promoter.
In a preferred ~ l - of the present invention, the HSV-I utilized
is deficient for expression of the thymidine kinase (lX) gene locus (TK(-) HSV-I).
More preferably, the G protein linked receptor sequence is inserted in the thyriudine
25 kinase (-IK) gene locus of the HSV-I genome, rendering it deficient. Within the
context of the pres~nt invention, "deficient" refers to low or ~ expression of
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the gene in question. Deficient expression generally results from insertion into or
deletion of the genetic loci in question. Deficiency of the thymidine kinase loci can be
assayed using any one of several means, including selection with blulllùd.,v~y~,Lillillc
using standard methods.
5 In another preferred rllll.O.i;".. ; of the present invention, the HSV-I
genome is deficient for the expression of virion host shut off gene (UL4 1 ) locus and the
thymidine kinase (TK) gene locus. Even more preferably, a nucleic acid segment
encoding bet~ r is inserted in the virion host shut-off gene (UL41) locus to
allow for easy . . " .1~. l ., - ;. ., l of successful ~ hilit~ti~n and the G protein linked receptor
10 sequence is inserted in the thymidine kinase (TK) gene locus. The deficiency in UL41
expression may be assayed for by detecting beta~ rt~iris~ expression using
standard techniques.
In another aspect of the present invention, the HSV-I genome is
additionally deficient in the expression of a viral gene required for replication
15 ("Ic~ ,aLiull deficient"). Briefly, proteins required for replication include, by way of
example, ICP4 and DNA polymerase. Preferably, it is replication deficient in theexpression of the ICP4 protein. Rl~rlit s~ti~ n deficiency can be assayed using any one of
several standard methods, including by C-- I~ of cultures in ..."l.l. .". .,:- y and
y cell lines.
In another cLubodilll~ L of the present invention, HSV-I is provided
which is both replication deficient and deficient in the expression of a viral host shut off
gene (UL41) locus. Even more preferably, it is deficient in the expression of both
UL41 loci and ICP4 protein.
Within the context of the present mvention, "vTKhml-l" refers to a
Ir~ HSV- I which is deficient in both the expression of the viral host shut off
protein (VHS) and thymidine kinase (-IK). (FIG. Ib); "vTKhml-2" refers to a
HSV-I which is deficient in the expression of both the viral ~
regulator (ICP4) and thymidine kinase (TK). (FIG. Ic); and "vTKhml-3" refers to a
1~ ,",1.;,.,,.,1 HSV-I whichisdeficientinboththeexpressionoftheviral ~
regulator, ICP4, VHS, and thymidime kinase (TK). (FIG. Id). All three of the
l~.. ,.,l. - -.,l viruses express a G protein linked receptor (preferably inserted in the TK
locus) from an immediate early promoter, preferably a CMV promoter. As described in
- more detail below, these 1~ ." .l .; . ,~ .I HSV-I are .. I, -- ,.. I rl; ~. .I by low uy ~up~lLllic;Ly and
a high rate of expression. Rcc..."l,;,.,..ll HSV-I viruses with "essentially the same
.. l,~.,.. ;~l;.~" is intended to refer to 1~ HSV-I with the same or similardr~-;. .1~; ~ inexpression.
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These and other 1~ ""1,;,.- ll HSV~ f. . ;~. .1 by low ~;y~ulJ~Llfi~,iLy
and/or a high level of expression of G protein linked receptor may be produced by
culturing a frst and second l,~.,..,l,;" ..l HSV-I in a suitable cell line for a time
sufficient and umder suitable conditions to allow for ,~ The first
5 l~ HSV-I is one carrying a G protein linked receptor gene amd capable of
expression thereof a~d the second ll ~I..l,11;11A.II HSV-I is replication deficient.
The G protein linked receptor nucleic acid segment may be inserted mto
the first .~. ,. . ,l,;",. ,l HSV-I by any suitable meams described above, including
homologous .~..,.,.1.;., ~;".. between the virus amd a plasmid carrying the G protein
10 linked receptor nucleic acid segment. ~ ""l,~ -,l HSV-I carrying the G protein
linked receptor sequence may then be selected for using st_nd~rd methods, including
restriction digestio l followed by Southem Blot hybridi~ation. Preferably, the first
,l HSV-] is TK(-) HSV-I. Even more preferably, the G protein linked
receptor gene is inscrted in the TK locus of the first .~ ",.l .;.,- ,l HSV-I . Additionally,
15 the first 1C~.III.I.;~IA.I HSV-I is preferably deficient in expression in the virion host shut-
offprotein (VHS). ~ost preferably, the first l~l ' virus is vhsA (available fromJ. Smiley, McMast r University, Hamilton Ontario) (FIG. Ia). Briefly, vhsA is a
mutimt HSV-I which bears the betA "OI- ~V~ gene in the UL41 region of its
genome, rendering it deficient in expression of the virion host shut-off protein. The G
20 protein linked recep-.or gene may be inserted into vhsA by the means described above.
Preferably, the second lr~ l HSV-I is replication deficient. Even
more preferably the second .,...,.,l.;",.,.l HSV-I is deficient in the expression of the
ICP4 protein. Most preferably, the second l~' ..,.,I.;,.,",i HSV-I is dl20. (Disclosed in
detail in DeLuca et al., "Isolation and (~ of Deletion Mutimts of Herpes
25 Simplex Virus Type I in Gene Encodmg Immediate Early Regulatory Protein ICP4," J.
of ViroL ~6:558-570 (1985)). Briefly, dl20 is replication deficient HSV-I, due to
diminished expression of ICP4. R~ f~ defective for ICP4 expression may be
selected using any one of several suitable methods noted above including Southern blot
analysis, Northern blot analysis, or i. "".." ,. . n. .. ~ studies.
30 If bcth the first and the second lc~ .. "h;lA.l HSV-I are replication
deficient, the t~vo l~ ....l.;. A,.~ HSV-I can be transfected on a ~ I A Y cell line
for replication. Su table ~"",l,! " ~ Y cell lines include E5 Vero cells (ICP4(+)).
(Disclosed in detail in DeLuca etal., "Isolation and ('IIA~ of Deletion
Mut~mts of Herpes Simplex Virus Type I in Gene Encoding Immediate Early
35 Regulatory Protein CP4,'l J. of ~irol. ~6:558-570 (1985)).
SUBSrlTUTE SHEET (FIULE 263
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2 1 76284
The ~ HSV-I resulting from the ~ ;.." of the first and
second Irl,~ l HSV-I are selected for one or more of four basic ~,II.. rl .. .;~
(I) thymidine kinase deficiency, (2) ICP4 expression, (3) UL41 expression. and (4) G
protein receptor gene expression, using any one of seYeral suitable methods described
5 above. By way of example, thymidine kinase expression can be screened for using
bloluod~w~y~.y~illiul~ ICP4 expression can be screened for based on the virus' ability or
inability to grow on the ~ . .". .,l;",g cell lines; UL41 expression can be screened for
based on beta-~,.l- I..~i.l,.~. ~JIUdU~ /ll, and expression of the G protein linked receptor
gene can be screened for based a on rihn~ ~lP~ protection assay. Maniatis et al.,
10 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1982). Thus, three preferred ~..,.1)~.1;..,..,1~ of the invention
vTKhml-l (FIG. Ib), vTKhml-2 (FIG. Ic), amd vT~hml-3 (FIG. Id) may be produced
and screened according to expression. The more preferred . ,..l.o~ ..1 is vTKhml-3
(FIG. Id).
As noted above, within other aspects of the present invention,
.l HSV-I can be used to deliver G protein linked receptor nucleic acid
sequence to ~llA~llll~rl;r~ cells. Once infected, the l~ HSV-I will then
produce the desired receptors which are expressed on the cell surface. The infected
cells are then selected for the desired G protein lirlked receptor expression. For virus
20 infection, the 1~ , l HSV-I may be applied to the cells umder standard cell
culttlre conditions. Cell culture techniques are described in Maniatis et al., Molecular
Clomng, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor.
N.Y. (1982). The specific host cells employed in the present invention are not critical
as long as they allow replication and expression of the l~' .. 1.;.. ,.. 1 HSV-I. Suitable
cells include Vero cells (ATCC Accession No. CRLI 587).
To select for the expression of G protern linked receptors, standard
techniques may be employed, including, ;1.1..l.l. l. -~. protection assays such as those
described in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Briefly, a labeled RNA probe is
30 tiyll~ that is pattially c.. ll.l,l.. 1- y to the region of the target mRNA. This
- labeled RNA probe is added to samples of the total cellular RNAs isolated from the cell
culttlre after post infection by the l~ virus. The mixture is incubated, for a
- sufficient time and under suitable conditions to enable a labeled probe to hybridize to
tbe ~ -'Y RNAs and then subjected to digestion by suitable restriction
35 enzymes, such as RNase A and RNase Tl. Labeled probe that hybridized to
SUBSTllllrE SHEET (P~ULE 26~
WO 95/13391 2 ~ 7 6 2 8 4 PCI'/IJS94/12996
. ~ ~
16
c..".~ y transcripts will be protected from digestion and may be separated on a
pOl~a~l ~la~ lC gel and viewed by ~
G linked protem receptor expression resultmg from tne insertion of the
1'~..1~.1,:..,.,.1 viruses of the instant invention into tbe cell can be detected using any one
5 of several methods known in tbe art, including for example, ligand bindmg assays.
R.,~Jlc~cllLaLive ligand binding assays suitable for use witbin tne present invention
include tnose descri ~ed in Conn, Methods in N~u, U.~ r,~ (VOl. 9)~ "Gene Expression
mNeural Tissues" Academic Press, Inc., San Diego, California (1992). For example,
witbin one rll,l.o~l . .,l the cells infected witb the l'' .. l. - ,l virus are incubated with
10 a rArl~ hPllPtl antagorlist. Saturation curves may tben be performed in order to
detcrmine the a~Jlu/~illl...~, number of receptors (lq~ .d by coumts measured using
tbe antagonist and ~ulllp~,-iLive inhibition). Witbin otber rll.l.O.l;..,...;~, stimulation of
second messenger sJstems maybe be ascertained by any one of several suitable means,
including, for example, pllu~llaiillyli~ ~l (Pl) turnover assays.
The l~ .. ,.1;, --,1 viruses of tbe present invention may be ~l --,.. ;~, ;,. I
in a variety of manners, including for example, by tbe number of receptors expressed on
cells infected witb -he virus, or the in vivo cyLu~Jailli.,;Ly of the virus. For example,
within certain r.l,lll,, l;.,... :~ of the present mvention, .ri~..."l.;.. ,l HSV-I are provided
which express grealer than 10,000 receptors per cell, typically an expression rate of
20 about between 25,000-200,000 receptors per cell, preferably an expression rate greater
than about 200,000 receptors per cell. Witbin other ....l.o~ , . ' ' viruses
are provided which have an in vivo cyLu,uailfi~ y of generally less tham the in vitro
eyL~ailll-,;Ly. ''Cylu,uaLL~,;Ly'' as used herein, refers to cell survival five days after
infection. CyLupaLII~,;ly may be measured usmg any one of a wide variety of
25 techniques known n the art, including ~.",."..,~lly available kits. Suitable kits
include Live/DeadTM (Molecular Probes Inc.; viability/~,yLuLu~i.,;Ly kit utilizing a
method of staining).
By way of example, vTKhml-l is ~ l. --,.. ~ ;,. ~l by a surface receptor
expression rate generally greater than lO,ûO0 receptors per cell; typically in about the
30 range of 60,000 to 80,000; and preferably in about the range of 70,000 to 100,000.
vTK~ml-2 is ~,1,-,.. .,;,.~1 by a cyL~ailliu;iy of generally less than
50%; typically in about the range of 35%~0%; and preferably in about the range of
20%-35%. vTKhm] -2 is further . l .,.. ,.. ..; ,. ~ by surface receptor expression generally
greater tham 80,000; typically m about the range of 120,000 - 160,000; and preferably in
35 about the range of 1~ 0,000 to 200,000.
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vTKhml-3 is ~ I by a ~;yLu~ailu~ y of about in vilro
l;ylU,uaiL~ y of less than about 3%, typically in about the range of 0.1% to 1.0% and
preferably in about the range of .001% - 0.1%. vTKhml-3 is further ..l,~ ,;,. .1 by
surface recepto} expression generally greater than 800,000 receptors, typically in the
5 ramge of 1-1.5 million receptors, and preferably 1.25-2 million ~c~ ul~cll.
In another aspect of the present invention, and using the techniques
described above, the l'' . ., . ,1,; ",.. ,l viruses of the present invention can also be packaged
in a suitable cell line. For example, within one ~.",l.o.~: "~ ,l of the invention,
l HSV-I is cultured ex vivo in suitable ,, - ...,.,.~ . cells. These cells may
10 then be introduced in vivo, using the techniques describe below, ie., ~ ruL ,L;~
;~,lu;ll;~ ;ull, for treatment of neurological disorders or analysis. Alt~,lllalivrl,Y~ the
l~.",.l,;"~ HSV-I may be introduced directly in vivo by any one of several methods
described below.
In another aspect of the present invention, the Ir~llllll,.~ ll viruses
15 described above are d~Lll;lua~,lcd to a mammal for the treatment of neuronal cell
disorders, in both the central and peripheral nervous system. Such viruses may be
utilized in tne treatment of a wide variety of disorders, including fo} example, brain
tumors, dc~ ;Yc disorders, neural disorders . l..,.. '.. ;,. ~l by abnormal gene expression, amd inberited disorders caused by a known gene defect.
The IC' - ~ viruses of the present invention may also be utilized to
deliver norrnal genes to affected genes. This allows for the treatment of deficiency state
disorders, usually of enzymes, by increasing production thereo Additionally, the
l,~.".,l.;,,~.,l virus can be used to decrease the production thereof by using antisense
sequences. This is useful in creating animal models for the deficiency disorders or
treating over expressive disorders.
The l,...,...l.~ ,l viruses of the present invention can be used to create
;1 state disorders involving structural or regulatory proteins, in a model
system, which could be used in efforts to establish and study methods of ~u.."~..,.. l;..
the effect of the imbalance.
In one aspect of the present invention, the IC~'''.'Il;'l ~I virus may be
- used to treat ~ lu,~ ., disorders including, by way of examples, Parkinsons
disease, Senile dementia, Cil,.l..,`.;l.~l cerebral atrophy, Hl~ lla chorea,
CclclJlu~,~,lclJ~,lla~ ll. L~ ;.... Amaurotic family idiocy, Lcu~udyaLIu,ully, Familial
myoclonus epilepsy, Hallervorden-Spatz disease, Wilson's disease, l.. IJ--~I ..1;...~1,.,
35 t~ ;..,., Westphal-Strumpell ~ uala, Paralysis agitans, Dystonia
Illua~ u~ulll deformans, torsion dystonia, Hallervorden-Spatz disease, Spasmodic
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18
torticollis, Cerebellar rlF~ ., "1,..,,~ SIY~l~vc~,leb~llar ~1~ ~,..,..,.1;""~ Friedrich's ataxia,
Marie's hereditary ataxia, A~ ULIU~ lateral sclerosis, Progressive muscular atrophy,
P~u~y~ ;ve bulbar palsy, Primary lateral sclerosis, Werdrlig-Hoffrnann disease,
Wohlfart-Kugelber~--Welander syndrome, Hereditary spastic paraplegia, Plu~,.,c.;ve
5 neural muscular atrophy, Peroneal muscular atrophy (Charcot-Marie-Tooth)
H~ v~, interstitial neuropathy (Dejerine-Sottas), Leber's disease, retinitis
;g~ A and fra-ile X disorder.
Ll another aspect of the present invention, l,....,.1.~ viruses may be
used to treat disoIders . ~ by abnormal gene expression, and inherited
10 disorders caused b~ a known gene defect. In addition to a number of the disorders
listed above, genes for defective enzymes have been identified, by way of exampie, for
(I) Iysosomal stora-e disorders such as those involving 13-11. ~.~-...;..;1-~ (Kornerluk
etal., J Biol Chem. 261:8407-8413 (1986); Myerowitz etal., Proc, NatL ~cad ScL
(USA) 82:5442-544 (1985); Tsuji et al., N. Engl. J. Med 316:570-575 (1987)), (2) for
5 .1. .. ;. .r;- ~ in II~U~ ~IIII;IIC ~ h~.~yl transferase activity (the "Lesch-Nyhan"
syndrome; Stout ~tal., Met. Enzymol. ISI:519-530 (1987)), (3) for arnyloid
pol~ ,~u~ ,;, (prealbumin; Sasaki et al., Biochem. Biophys. Res. Cornmun. 125:636-
642 (1984)), (4) fcr Alzheimer (amyloid Tanzi etal., Science 235:880-884 (1987);Goldgaber et al., Science 235:877-880 (1986)), (5) for Ducherme's muscular dystrophy
20 (~ muscle protein; Monaco etal., Nature 323:646-650 (1987)), and
(6) for ~ r~blA~ - .,- l protem expressed in the retina and other tissues,
Lee et al., Science 235:1394-1399 (1987); Friend et al., Nalure 323:643-646 (1986)).
Rc( ..l,l.;.. l viruses may also be used to study the "shiverer" mutdtion
(myelin basic protein, Roach etal., Cell 42:149-155 (1987); Molineaux etal., Proc.
25 Natl. Acad Sci. (USA) 83:7542-7546 (1986), and the "jumpy" mutation
(proteolipoprotein, ~ave etal., Proc. Natl. Acad Sci. (USA) 83:9264-9268 (1986);Hudson et al., Proc Natl. Acad Sci. (USA) 84:1454-1458 (1987)).
Rf~ viruses of the present invention can also be used for
treatment of acute injuries to the brain or peripheral nervous tissue, for example from a
30 stroke, brain injury, or spinal cord injury.
Rr. .~ viruses of the present invention may also be used in the
treatment of disor~ers which require receptor mnrl~lu1inn to increase or decrease
transmitter upt. ke. Such disorders include ~ u~l., II;d, obsessive-.v...~ ;v~
disorder, depression, and bipolar mood disorders.
As utilized within the context of the present invention, the term
"treatment" refers to reducing or alleviating symptoms in a subject, preventmg
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symptoms from worsening or ,UlV~ .lllg5, inhibition or elimination of the causatiYe
agent, or prevention of the infection or disorder in a subject who is free therefrom.
Thus, for example, treatment of infection includes destruction of the infecting agent,
inhibition of or illt~.r.l.ll.~ with its growth or mq~nrqtiAn l~ ., of its
5 rqthAl~ iAql effects and the like. A disorder is "treated" by partially or wholly
remedying the deficiency which causes the deficiency or which makes it more severe.
An unbalanced state disorder is "treated" by partial~y or wholly remedying the
imbalance which causes the disorder or which makes it more severe.
~rhe .c~ l viruses of the present invention may be adl.l;lll~
10 by any one of several methods of A~ known in the art which account for the
risk of ~ of the IC~ I virus in the bloodstream and such that the virus
retains its structure and is capable of infecting target cells. Within one rmhoflimPnt
a.l...;.,.~ .... may be A~....,.l.li~l.~d by lll;.,luill;c.~iull of the virus, alone or in a
1; Ally suitable carrier or diluent~ through a ~Ll~iula.,li.,dlly-located pipette or
15 syringe. Suitable locations vary with Arplirqti~An; but include intraocular and brain
injections.
rl ~ I carriers amd diluents which are suitable for use within the
present invention include, for example, water, lactose, starch, ..,^~ ;..,.. stearate, talc,
gum arabic, gelatine, uul~alhyl~ . glycols, and the like. The l~
20 ,ul~pala~ivll may be made up in liquid form for example, as solution, emulsion,
suspension and the like or in a solid form, for example as a powder and the like.
If necessary, the l.l.~.",,-~..l;.Al preparations can be subjected to
UUII~ iUIIdI ~ adjuvants such as preservmg agents, stabilizing agents,
wetting agents, salts for varying the osmotic pressure, and the like. The present
25 ~ UICIJalaLiVI~ ~ may also contain other ~ lly valuable substances.
In another aspect of the present invention, .. ' viruses may be
delivered by chronic irlfusion using amy suitable method known in the art, including an
osmotic minipump (Alza Corp.) or delivery through a time release or sustained release
medium. Suitable time release or sustained release systems include any methods
30 known in the art, including media such as Elvax (or see, for example, U.S. Patent Nos.
5,015,479, 4,088,798, 4,178,361, and 4,145,408). When using chronic infusion, time
release, or sustained release ,.l. . l. .l:~.l,c the lc~ .~.,l .;.l- ll virus ~...1ll1.l .~;l;..l. may be
injected into the c~ blv ~pillàl fluid via intrathecal or illLIa~ ,ulal injections, as well
as into the brain substances and mtraocular locatiorLc.
The ~ virus should be d~ l in a ~ AIIY
effective amount. A 11l~ AIIY effective amoumt is that sufficient to treat the
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disorder. A ~ y effective amount can be determined by in vitro experiment
followed by in vivo studies. Expression of the inserted nucleic acid segment can be
deter~nined in vitro siag any one of the techniques described above. Expression of the
inserted nucleic acid segment can be determined in vivo using any one of several5 methods known in the att, including i."""."..n"..,~ cc using a nuul~ J
ligand.
In another aspect of the present invention, the ~.1.",l,;.,-.,l HSV-I
viruses described above are iulCul~ ' into a 1111AIIIIA- ~- ;;1 AI CU~ UU~ UII~
Preferably, the II~ AI CU..,~ contains one or more II~ .II;.AIIY
10 effective dûses of the l- ~ -,-,l-; ---.l virus in a suitable l.l.A-" ~ ;- Ai carrier or diluent.
Suitable ~ A.IIIA...II;.AI carriers and diluents are outlined above. A th~n~r~l.tiA~IIy
effective dose may be determined by in vitro experiment followed by in vivo studies as
described above. The i.,.",l.~.~;l;..,l may be a.lll,ulia~tl~d by any one of the methods
described above.
The f~llowing examples are provided by way of illustration, and not by
way of limitation. Unless otherwise indicated, the specific protocols used in the
following examples are described ia detail in Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory (1982).
EXAMPLE 1
GENERATION OF AN HSV-I RECOMBINANT ~ lNG ml
MUSCARINIC ACETYLCHOLINE RECEPTORS
A e~--"-l,'--,l HSV-I virus which expresses the ml muscarinic
ac. .yliLoliae recep-or (ml-AchR) was generated by llulllolo~ uua ~
between an HSV-I virus and a plasmid, pTKhml, which was constructed for this
purpose.
Briefly, pT~hml was prepared from the coding sequence for the human
ml-AchR gene and altered pTKSB. The coding sequence of ml-AchR was isolated as
a 2 7 kb BamHI fra~~ment from a starting plasmid supplied by Bonner (Laboratory of
Cell Biology, National Institute of Mental Health, Bethesda, Marylaad) arld inserted
iato a plasmid vectol containiag a siagle BarnHI cloning site The codiag sequence was
re-isolated by digestion of that plasmid vector with EcoRI aad Hin~lJ
pTKSB (available from J Smiley, McMaster Universit~v, Hamilton
Ontario; Smiley et al., ~ Virol. 61(8):2368-77 (1987)), which contains the HSV-I TK
gene, was altered by insertion of a CMV promoter-containing fragment from the
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21
plasmid pRc/CMV (Invitrogen Corporation). This fragment represents the portion of
the plasmid extending from base 209 to base 1285 and containing the CMV major
immediate early promoter, a, . ~ site, and a poly A addition site. The fragment
was inserted into pTKSB by first digesting the plasmid with BamHI and then
5 converting the BamHI site into a PacI site by the addition of adapter sequences. The
CMV promoter was oriented in the opposite direction to the TK promoter to reducetranc~irti--n~ . The resulting plasmid (pTKSB containing the CMV
promoter) was then digested with EcoRI and HindJlI and ligated to the ml-AchR
coding sequence which had also been digested with HindlII and EcoRI usimg
10 conventional methods. This plasmid was referred to as pTKhm I .
pTKhml was then used to generate an HSV l~ ".,l.;,._.,~ virus by in vi~o
homologous ~ ;..l. pTKhrnl was ~ f~ ~ lrd into Vero cells (ATCC
Accession No. CRL1587) along with an infectious HSV-I, vhsA. vhsA is a mutant
HSV-I (FIG. Ia) (available from J. Smiley, McMaster University, Hamilton Ontario)
15 containing the ~ rtnci~l~c~ gene in the UL41 gene coding sequence.
TK deficient l~....,l,;,.-.~ were selected using blvlllou~,u~y~y~idille.
Following selection, virus isolates were plaque purified and tested for the CMV-ml-
AchR insert by digestion with EcoRI, el~LIuL,l.vl~, ,;, on a 1.1% agarose/TAE gel and
l~ylvlhli~Livll to a radioactive probe. The probe was generated by incubating the
20 mlAchr gene in buffer containing random hexamers of DNA to act as primers forextension by DNA polymerase in the presence of dGTP, dTTP, dATP, and 100 mCi
[32p] dCTP. After 3 h of incubation. the probe was used in Il~I,lhli ,.Liull at 37C in the
presence of 50% f~-rmami~lf 2X standard saline citrate, 5X Denhardt's solution, 1%
sodium dodecyl sulfate. Following incubation for 12 h, filters were washed ~ ai~ ~
25 in 0.2 X SSC, 0.1% SDS, dried, and exposed to X-ray film until a signal was detected.
One virus, referred to as vTkhml (FIG. Ib), lacked a 2.1 kb EcoRI fragment containing
the ..l,l~ ...."~ TK gene and instead, contained a 4.6 kb EcoRI fragment which
hybridized to the ml-AchR specific probe. Thus, it was ~ t~rmin( d that the
ll~,~uLIallall~i~l receptor gene was successfully introduced into the viral genome.
EXAMPLE 2
DETECTlON OF mlACHR mRNA EXP~ESSION FROM
- RECOMBINANT VIRUSES
F~pression of mlAchR transcripts from the CMV promoter was detected
using a ~ 5 r protection assay. (FIG. 2). A labeled RNA probe was a.yllLl
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from 326 l~u~ o~id~ (nt) from the T7 promoter of the plasmid BS/KS(-) (availablefrom Stratagene Cloning Systems) comprising 265 nt of the 5' end of the mlAchr gene
and 56 nt of the 3' end of the CMV promoter. This probe targeted the 5' end of humam
mlAchR mRNA as well as a portion of the CMV promoter. This labeled probe was
5 incubated with sam~les of total cellular RNAs isolated from Vero cells 2 to 18 hours
post-infection (hpi) ~y vTKhml.
The Ieaction was then subjected to digestion by RNaseA and RNaseTI
under conditions of ligh salt to inhibit digestion of double-strand RNA. Labeled probe
that had hybridiæd to cellular RNA was ~ .. ly identified following
10 cl~ -uullu~c,;~ on an 8M urea/polya~lyLl-ide gel and visualized by ~ y
(FIG. 3). A protected RNA fragment of 265 nt which l,u~ ùll ;Ic ;I to Llall~ iull of
the insert from the CMV promoter was detected as early as 3 hours post-infection("hpi"), reached high levels by 8 hpi, and mamtained high levels until 18 hpi. (FIG. 3).
EXAMPLE3
ISOLATION OF ICP4-RECOMBINANTS EXPRESSING THE mlACHR GENE
Rcc~llllll;l.r.l;~ were generated by IIUIIIOIO~UU~ II between
two viruses: dl20, an ICP4(-) virus developed by DeLuca, (DeLuca et al., "Isolation
20 and (81lA.... ~1 .;~A;;I~n of Deletion Mutants of Herpes Simplex Virus Type I in Gene
Encoding Immediate Early Regulatory Protein ICP4," J. of l~irol. 56:558-570 (1985)),
and vTKhml (FIG. Ib), prepared in Exatnple 1. Briefly, the viruses were coinfected
with E5 cells, an ICP4-expressing Vero cell line. The resulting virus stock was selected
for TK(-) mutants with blulllùd~,u~y~,y~;dill~, and clones were screened for their ability
25 to grow on E5 cells, ~ut not Vero cells.
Positive clones were then tested for the presence of the mlAchR gene by
restriction digestion with EcoRI and Southem blot llybl;diLaliull. One virus clone,
referred to as vTKhml-2 (FIG. Ic), was found to both express mlAchR and fomm
plaques only with E5 cells.
This IC~ ".. ,i ,., - - ,I was then used to generate a third ~c c .. 1,; "A . ~1 referred
to as vTKhml-3 (FIG. Id), which is defective in both ICP4 and VHS expression. E5cells were coinfected with vTKhml-2 (FIG. Ic) and vhsA, the HSV-I mutant that
expresses ~ from its UL41 region. B~u~l~ùdcu~y~,y~idill~ was used to
select against vhsA, land the resulting viral isolates were screened (a) for their ability to
35 grow on E5 cells, but not Vero cells, (b) for the expression of mlAchRs, and (c) for the
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expression of ~ rtrci~ q These lc~ were referred to as vTKhml-3
(FIG. Id).
EXAMPLE 4
DETECTION OF SURFACE RECEPTOR EXPRESSION FROM
RECOMBINANT VIRUSES IN VERO CELLS USING LIGAND
BINDING ASSAYS
The expression of mlAchR from Vero cells infected with a lll~lLi~ y
of infection of 10 with vTKhml-l, vTKhml-2 and vTKhml-3 was compared using the
[3H]NMS ligand binding assay. Surface mlAchR were measured by incubating
infected Vero cells witb I nM of the n~lir,~ muscarinic receptor antagonist, n-
methyl-a~uyulalll;llc ([3H]NMS) at 37C for I hour. After incubation witn [3H]NMS~
the infected cells were washed three times with phosphate buffered saline, Iysed amd
counted in srintillqti~n fluid. Saturation curves were performed to determine the
apylu;~ , number of mlAcbRs lc~ lt~,~ by coumts measured usmg InM
[3H]NMS. (FIG. 4). Competitive inhibition by y;lc~ J;Il., confirmed that these
coumts reflect specific binding of the ligand to mlAchRs.
Vero cells do not contain any rll.ll~ mlAchRs, therefore any
[3H]NMS binding above b~ represent receptors expressed from the
IC~ virus. The expression of mlAchRs from each lc- .."l.;.,- " is shown.
(FIG. 4). The ICP4-mutant, vTKhml-2 infected Vero cells exprcssed 2-3 fold more
mlAchRs than the VHS-mutant, vTKhml-l infected Vero cells. Vero cells infected
with the triple mutant, vTKhml-3, expressed greater than 5-fold more receptors than
25 those infected with vTKhml-l and at least 2-fold more tban those infected with
vTKhml-2 in the first 12 hours following infection. After 20 hpi, mlAchR surfaceexpression appears to plateau. At 36 hpi mlAchR surface expression from vTKhml-2and vTKhml-3 arc ~.y~ J the same. Receptor expression from vTKhml
plateaus by aylJlwdilll~t~ 12 hpi, and by 36 hpi Vero cells infected with the replication
30 competent vTKhml-l l~ are dead.
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EXAMPLE 5
DETECTION OF SURFACE RECEPTOR EXPRESSION FROM
RECOMBINANT ~'IRUSES IN E5 CELLS USING LIGAND BINDING ASSAYS
The expression of mlAchR from E5 cells, ICP4(-) Vero cells, infected
with a lllu'~lh,;iy o `infection of 10 with vTKhml-l, vTKhml-2 and vTKhml-3 was
compared using the 3ame [3H]NMS ligand binding assay as im Example 4. (FIG. 5).
of the ICP4(-) mutation in vTKhml-2 and vTKhm1-3 transfected E5
cells results in drastically reduced levels of mlAchRs. (FIG. 5). These results indicate
that the increased expression levels in vTKhml-2 and vTKhml-3 infected Vero cells is
related to lack of ICP4 expression. The expression of ICP4 by the E5 cells allows the
'' . " "h; "~, .i viruses ~o replicate. (FIG. I l). This data further indicates that the lack of
viral host-protein s ~nthesis (VHS) expression contributes to increased mlAchR
expression, since v~Khml-l and vTKhml-3 have higher expression levels than
vTKhml-2 in E5 cells.
At I hpi and 12 hpi DNA was isolated from each of the infected Vero
and E5 cell samples ~y standard methods and dotted onto nitrocellulose membrane in
three fold dilutions. (FIG. I l) vhsA infected Vero and E5 cell samples served as a
control. These resultj .1. .,.1.,.~l...~. that vTKhml-2 and vTKhml-3 samples replicated
20 in the E5 cell samples, but not in the Vero cell samples.
EXAMPLE 6
CONFIRMATION OF DEFECTIVE ICP4 EXPRESSION IN vTKhml-2
AND vTKhml-3
Southern blot analysis amd ;.... ~ u,a""l~,f studies were performed
to ensure that the l~ vTKhml-2 and vTKhml-3, were defective in ICP4
expression. Southern blot confirmed the presence of a 4.05 kb deletion in ICP4. This
deletion is ..1. ,- ' ;~ of dl20, the ICP4(-) HSV-I strain used to construct these
30 r~ ....-.1.:.. .: ~ The expression of the ICP4 product in Vero cells infected with the HSV-
I l.jf.""l.;"A.. I~ was assayed by indirect ;,.. ".. ~ l"e using a ,.. ~
antibody directed aga nst ICP4. Fluul~ II., of Vero cells infected with
either (a) vTKhml-l, (b) vTKhml-2 or (c) vTKhm-3 at 4 hours post-infection were
produced. The ICP4 antigen could only be detected in Vero cells rnfected with
35 vTKhml-l; vTKhml-2 and vTKhml-3 infected Vero cells did not express detectable
amounts of ICP4. (FIu. 9).
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EXAMPLE 7
EXPRESSION OF mlACHRs FROM HSV-I RECOMBINANTS IN PRIMARY
CORTICAL NEURON CULTURES
Primary cortical neuron cultures, isolated from seven-day-old neonatal
rats, were infected with either vTKhml-1, vTKhml-2, or vhsA at a multiple of
infection of 3. At 12 hpi, the cultures were incubated at 37C with [3H]NMS for I
hour. In addition, uninfected control cultures were assayed to measure the amount of
Pn~io~Pntnl~ mlAchR expressed in prima~y cortical neuron cultures. Atropine, an
mlAchR antagonist which competes with [3HlNMS binding, was used to determine theamount of nnn~r~nifi~ ligand binding present in each sample. (FIG. 7).
In these assays, vTKhml-2 infected cells expressed 5 fold more
mlAchRs than uninfected cultures, or ~u~u~ ,ly 38,000 surface receptors per cellas compared to 6,000 receptors on an uninfeckd cell. (FIG. 6~. However, cells infected
with vTKhml-1 expressed less than a 2-fold increase in the amount of mlAchR
compared to uninfected cultures. (FIG. 6). vhsA infected cultures expressed fewer
receptors than the uninfected cultures. (FIG. 6). Moreover, there were no cytopathic
effects evident in either vTKhml-2 infected neurons or the vTKhml-l infected
neurons. These results '-- that the 1~..."1,;.,--,l viruses of the present
20 invention reduce cytopathic effects associated with viral infection and provide
heightened expression of nucleic acid segment inserts.
A phosphatidylinositol turnover assay was performed on neuronal cells
infected with each of vTKhml-l, vTKhml-2, and vTKhml-3. This assay ,irlll.~ r~
that the mlAchR function to stimulate second messenger systems. 10 d cultures of25 mouse cortical neurons were infected or mock-infected amd then incubated prior to
of PI turnover using 1 uCi/ml [3H] inositol in inositol-free minimal
essential medium. Cultures were washed 3X m Hanks buffered saline solution. Cells
were treated or mock-treated with I mM carbachol. After 45 min, the medium was
removed, cells were washed once with HBSS, cold 3% perchloric acid was added, and
30 inositol phosphate levels were determined exactly as described previously (Murphy
- et al., FASEB J. 4:1624-1633, 1990). Second .. ~ were stimulated 5 fold by 12
hpi in infected Vero cells. Second ,..~ were stimulated 4 fold in rat cortical
neurons.
From the foregoing, it will be evident that although specific
35 ~ .o.i;..,..,l~ of the invention have been described herein for the purposes of
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ctr~tirm various mQ~lifir~ti~nc may be made v~ithout deviating from the spirit and
scope of the invention.
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