CA2176284A1 - Method of treatment using, process of preparing, and composition comprising a recombinant hsv-1 - Google Patents

Abstract

Methods for treatment, processes for preparing, and compositions for delivering nucleic acid segments to non-mitotic cells, primarily of the treatment of neurological disorders and exploring neurological functions, are disclosed. In particular, the invention provides recombinant HSV-1 with a high rate of expression of foreign nucleic acid segments and or a low cytopathicity and its associated methods and processes.

Description

WO 95113391 , PCT/US94/12996 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~

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) ~ 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 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) wo gSrl3391 PCT/U594/12996 '"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 ~ - 2 ~ 76284 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~

.. ~ ; 21 762~4 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 SUBSTlTIJTE SH~ lUi E 26~

WO 95113391 i PCIIUS94112996 .`.~' i~ i`. 2~76284 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.
SU8STlllJTE SHEET (RULE 263 2 ~ 76284 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 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 . ~ ~

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.
SU~SrlTUTE SHEET (RULE 26 WO 95/13391 '~ . . PCT/US94112996 2 ~ 76284 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 SUBSTITUTE SHE~ lULE 26~

WO 95113391 2 1 7 6 2 ~ 4 PCT/US94/12996 , . .. .

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 SUEiSrlTUTE SHEEr (RULE 263 WO 95113391 `.;- - 2 ~ 7 6 2 8 4 PCIIUS94/12996 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 SUBSTITUTE SHEET (F'IULE 26) WO 95/13391 ~ 2 1 7 6 2 8 4 PCI/US94112996 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).

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 SUBSTITUTE SHEET (RULE 263 WO 95/13391 . ~ r~ 2 1 7 6 2 8 4 PCTIUS94/12996 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.

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 StJl~STlTUTE SHEI~ (RULE 26) wo 95/ 1 3 3 9 1 ,.~ 2 7 6 2 8 PCI IUS 9 4/ 1 2996 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).

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 StJSSTlTUTE SHEET ~IULE 26'~

WO 95113391 ~ ' , '` 2 1 7 6 2 8 4 PCI/US94/12996 expression of ~ rtrci~ q These lc~ were referred to as vTKhml-3 (FIG. Id).

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.
SLleiSTlTlJTE SHEET (RULE 26'~

WO95/13391 ,.~. '.,t`~ 21762~4 PCrlUS94/12~96 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.

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).
SU8~TITUTE SHEEr (RULE 26) ,, _ .

WO 95/13391 ' 2 1 7 6 2 8 4 PCIIUS94112996 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 SUBSrlTUTE SltEET (~ULE 26~

W0 95/13391 ` '` ~ ~ PCT/US94/12996 ctr~tirm various mQ~lifir~ti~nc may be made v~ithout deviating from the spirit and scope of the invention.

SUBSrlTUTE SHE~ (RULE 25

Claims (37)

1. A recombinant Herpes Simplex Virus-1 capable of directing the expression of a G protein linked receptor gene.

2. A recombinant Herpes Simplex Virus-1 capable of directing the expression of an antisense transcript of a G protein linked receptor gene.

3. The recombinant Herpes Simplex Virus-1 of claims 1 or 2 wherein said recombinant virus is deficient for thymidine kinase expression.

4. The recombinant Herpes Simplex Virus-1 of claim 3 wherein the gene encoding the G protein linked receptor or the antisense transcript is inserted in a TK locus of said recombinant virus.

5. The recombinant Herpes Simplex Virus-1 of claim 1 wherein the G-protein linked receptor gene is a human M1 muscarinic acetylcholine receptor gene.

6. The recombinant Herpes Simplex Virus-1 of claim 1 wherein the G
protein linked receptor gene is an adrenergic receptor.

7. The recombinant Herpes Simplex Virus-1 of claim 2 wherein the antisense transcript is that of a 5-HT2 receptor gene.

8. The recombinant Herpes Simplex Virus-1 of claims 1 or 2 wherein said virus is deficient in expression of a virion host shut-off protein (VHS).

9. The recombinant Herpes Simplex Virus-1 of claims 1 or 2 wherein said virus is replication defective.

10. The recombinant Herpes Simplex Virus-1 of claim 9 wherein said virus is deficient in expression of ICP4 protein.

11. A recombinant Herpes Simplex Virus-1 having essentially the same characteristics as vTKhml-1.

12. A recombinant Herpes Simplex Virus-1 having essentially the same characteristics as vTKhm1-2.

13. A recombinant Herpes Simplex Virus-1 having essentially the same characteristics as vTKhm1-3.

14. A method of treating mammals for neurological disorders. comprising administering to a mammal a composition comprising a recombinant HSV-1, according to any one of claims 1-13, in combination with a pharmaceutically acceptable carrier or diluent.

15. The method of claim 14 wherein said wherein said composition is administered by stereotactical microinjection.

16. The method of claim 14 wherein said composition is administered by a time release mechanism, a sustained release mechanism, or chronic infusion.

17. Ex vivo mammalian cells infected with a recombinant HSV-1 according to any one of claims 1-13.

18. A pharmaceutical composition comprising a recombinant virus according to any one of claims 1-13 and a pharmaceutically acceptable carrier or diluent.

19. A process of producing recombinant HSV-1 virus with low cytopathicity, comprising:
culturing mammalian cells with a first recombinant HSV-1 containing a G
protein linked receptor gene and a second recombinant HSV-1 defective in a gene required for replication under conditions and for a time sufficient to allow recombination of the first and second viruses; and, selecting a recombinant virus by detecting G protein linked receptor expression.

20. The process of claim 19 wherein said G protein linked receptor gene is inserted in the thymidine kinase locus.

21. The process of claim 20 wherein the G protein linked receptor gene is an adrenergic receptor gene.

22. The process of claim 20 wherein the G protein linked receptor gene is a human M1 muscarinic acetylcholine receptor gene.

23. The process of claim 19 wherein the first recombinant virus is deficient in the expression of the TK locus.

24. The process of claim 19 wherein the first recombinant is deficient for expression of the virion host shut-off protein VHS.

25. The process of claim 19 wherein the first recombinant virus is vhsA.

26. The process of claim 19 wherein the second recombinant virus is d120.

27. The process of claim 19 wherein the protein required for replication is ICP4.

28. The process of claim 19 wherein the recombinant virus is vTKhm1-1.

29. The process of claim 19 wherein the recombinant virus is vTKhm1-2.

30. The process of claim 19 wherein the recombinant virus is vTKhm1-3.

31. A recombinant HSV-1 with an in vitro cytopathicity generally less than about 3%.

32. The recombinant virus of claim 30 wherein the cytopathicity is in about the range of 0.1% to 1.0%.

33. The recombinant virus of claim 31 wherein the cytopathicity is in about the range of 0.001% to 0.1%.

34. A recombinant HSV-1 capable of directing the expression of a G
protein linked receptor, said HSV-1 being capable of expressing on the surface of an infected cell greater than 10.000 receptors/cell.

35. The virus of claim 34 capable of expressing on the surface of an infected cell about the range of 25,000-200,000 receptors/cell.

36. The virus of claim 34 capable of expressing on the surface of an infected cell about the range of 200,000 to 400,000 receptors/cell.

37. A recombinant Herpes Simplex Virus-1 according to any one of claims 1-13, for use in the manufacture of a medicament for treating mammals with neurological disorders.