CA2234604A1 - Recombinant herpes virus vectors for expression in neuronal cells - Google Patents
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Abstract
Methods for treatment, processes for preparing, and compositions for delivering selected nucleic acid sequences to cells, primarily of the treatment of neurological disorders and exploring neurological functions, are disclosed. In particular, the invention provides recombinant Herpesvirus vectors with a high rate of expression of selected nucleic acid sequences and/or a low cytopathicity and its associated methods and processes.
Description
WO 97/13866 ~CI'~rJS96~1636 Description RECOMBINANT HERPES VnRUS VECTORS FOR EXPRESSION nN NEURONAL CELLS
Technical Field The subject invention is generally directed to a recombinant Herpesvirus vectors and more specifically, recombinant Herpesvirus vectors with a high rate of expression of foreign gene sequences and/or a low cytopathicity in neuronal cells.
Back~round of the Invention The capacity to introduce genetic sequences into a m7~mm~ n cell and to 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 de~igning a therapeutic basis for the treatment of disease.
The introduction of a genetic sequence into a m~mm~ n host cell may be facilitated by first introducing the sequence into a suitable vector. However, vectors suitable for use in nonmitotic cells, such as neural or neuronal cells, has proven challenging. In addition, whereas most tissues in the body are readily zl~cf ~ihle 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-replicative state of most nerve cells, present peculiar challenges when ~le~ ning gene therapy systems.
These challenges have hindered the possible tre~tmenf of neurological disorders such as brain tumors, de~ne.dli~e disorders (multiple sclerosis, Parkinson's disorder, Alzheimer's disease, ~ y~Llophic lateral sclerosis), disorders caused by abnormal expression of genes, inherited disorders caused by a known gene defect (HPRT in Lesch-Nyhan disorder; retinblastoma ~Lee et al., Sci. 235:1394, 1987);
glucocerebrosidase (Sorge et al., Proc. Natl. Acad. Sci. USA 84:906, 1987); and Duchenne's muscular dy~lloplly (Monaco et al., Nature 321:443, 1986)) and acute injuries to the brain or peripheral nervous tissue, for exarnple from a stroke, brain injury, or spina} cord injury.
Although many viral vector systems have been developed, there has been difficulty adapting these systems for neuronal cells. For example, although retroviral vectors have been used to L~ l genes into neuronal cells in vitro (Price et al., Proc.
Natl. Acad. Sci USA 34:156-160, 1987), and in vivo ~Culver etal., Science 256:1550, 1992); Price et al., supra~, they have not proven useful in delivering genes to a large plupollion of cells in the nervous system. Other viral vector systems also have characteristics limiting their usefulness for gene transfer into neuronal cells, such as:
5 rapidly clearing Iytic infections (e.g, adenovirus~ vaccinia virus), small genome size (SV40, polyoma), or limited cell tropism (EBV, bovine papilloma virus).
A Herpes Simplex Virus-l (HSV-l) vector has been shown to be useful for infecting a wide variety of cells, including neuronal cells (Spear and Roizrnan, DN~
Tz~n20~ Viruses, Cold Spring Harbor Laboratory, NY, pp. 615-746). Briefly, HSV-I can 10 exist in a latent state in neural cells (Stevens, Microbiol. Rev. 53:318, 1989) allowing for m~inten~nce of the vector. Additionally, the viral genome of HSV-l is very large (150 kb) and may accommodate large nucleic acid segment~.
Geller et al. (PCT WO 90/09441) developed a HSV-l virus-based vector, which, while offering advantages over plasmid-based vectors, has failed to be 15 efficacious in several in~t~nres. These vectors suffer from low gene ~x~le3~ion and high cytopathicity, thus severely limi~ing their use in gene transfer. While others have tried to increase expression by using a variety of promoters (Tackney, et al, ~ Virol.
52:606, 1984), cytopathicity has been shown to be a persistent problem, even in those viral vectors which are replication deficient (Johnson etal., J. I~irol. 66:2952, 1992;
20 Johnson etal., Mol. Brain Res. 12:95, 1992). For long-term expression in neuronal cells, it is necessary to have a viral vector that demon~ll~es low cytopathicity.
In view of the inability of current HSV-I vectors to adequately account for the balance of cytopathicity and gene ~ ession, it is apparent that there exists a need for new and additional methods and compositions which address and rectify the problem.
25 The present invention fulfills this need, and further provides related advantages.
Summarv of the Invention Briefly stated, the present invention provides t;~ression ç~csett~s capable of e2~ ssillg a sequence of interest. Within one aspect such expression 30 c~sett.?s comprise one or more neuronal specific silencer elements, a promoter element operably linked to a sequence of interest, and an enhancer, wherein the enhancer and silencer elements are positioned such that they are not adjacent to one another.Within one embodiment, the silencer element is a neuronal restrictive silencer element, and the promoter element is selected from the group consisting of 35 CMV, SV40, herpes promoters and adenovirus promoters.
W O 97113866 PCT~US96/16368 Within other aspects, expression cassettes are provided a LAT promoter operably linked to a sequence of interest, followed by an enhancer. Within further embofiiment~, such e~p~ ion cassettes further comprise a neuronal-specific silencer element, wherein the enhancer and silencer elements are positioned on the vector such S that they are not adjacent to one another. Within further embotliment~, the expression cassettes are capable of expressing a sequence selected from the group consisting of antisense and ribo~yme sequences, sequences which encode disease-associated antigens, sequences which encode imrnunologically active molecules, replacement genes, arld toxic genes.
Within other aspects of the present invention, gene delivery construct, as well as host cells are provided which contain one of the above-identified expression ç~c~ett~s. Representative examples of suitable host cells include cells from a warm-blooded animal or vertebrate, and includes in particular neuronal cells such as cortical neurons, cerebellar granule cells, retinal ganglia cells, hippocampal neurons, peripheral 15 sensory neurons, and motor neurons.
Within another aspect of the present invention, methods are provided for producing a protein, comprising the steps of (a) introducing an e~res~ion cassette or ~ene delivery construct as described herein into a host cell, and (b) cultllring the host cell under conditions, and for a time sufficient, to permit ~ ion of the protein.
20 Within further embo-liment~, such methods further comprise the step of purifying the protein.
Within other aspects of the present invention, methods are provided for introducing a selected se~luence of interest into neuronal cells of a warm-blooded animal, comprising the step of ~lmini~tering to said animal an expression ~ ettP or 25 gene delivery construct provided herein. Such vectors may be ~flmini~tered by a variety of routes, including for example, subcutaneously, intracranially, intradermally,intr~mll~c~ rly, hllla~ oneally, or intravenously. Within other embot1imt~.nt~, the vector may be directly ~imini~tered to a tumor.
Within other aspects, methods are provided for introducing a selected 30 sequence of interest into an in vitro culture cont~ining neuronal cells, compri~ing the step of introducing an expression cassette or gene delivery construct as described herein ~ into an in vitro culture cont~ining neuronal cells.
The present invention also provides recombinant Herpesvirus vectors capable of directing expression of a C~ protein linked receptor gene. Within certain 35 embo~liment~ of the invention, the recombinant viruses direct the expression of such CA 02234604 l998-04-l4 W O 97/13866 PCTrUS96/16368 genes in non-mitotic m~mm~ n cells, and more preferably, in m~mmz~ n neuronal cells.
Within other aspects of the present invention, recombinant Herpesvirus vectors are provided which are capable of directing the c;x~es~ion of an antisense 5 transcript of the G protein linked receptor gene.
In one embodiment of the invention, recombinant Herpesvirus vectors 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 embodiment of the present invention, the gene encoding a G
protein linked receptor or a antisense segm~nt thereof is inserted in the TK locus of a Herpesvirus genome. For example, the antisense se~ment may be a ~-HT2 receptor gene. Numerous G-protein linked receptor genes may be utilized within the context of the present invention, including, by way of example, a human Ml mn~c~rinic acetylcholine receptor gene or an adrenergic receptor.
Within other aspects of the invention, methods of treating m:~mm~l~ for neurological disorders are provided, com~ri~in~ the step of ~mini~tering to a m~mm~l a composition comprising an expression cassette or gene delivery construct such as a recombinant Herpesvirus vector, as described above. Within certain embodiments, this may be accomplished in combination with a phs~rm~ce~ltically acceptable carrier or diluent.
Within certain embo-lim~nt.c, the ~-1mini~tration of pharmaceutical compositions may be accomplished by, for example, by stereotactically microinjection, a time release mech~ni.cm, a sustained release merh:~ni.~m, chronic infusion, or ex vivo m~rnm~ n cells infected with or c~r~ a recombinant Herpesvirus vector~ gene delivery constructs, or expression cassette according to the present invention.
Another aspect of the present invention provides ph~rmzlrelltical compositions comprising an expression cassette or gene delivery construct of thepresent invention and a ph~rm~- eutically acceptable carrier or diluent.
Within yet other aspects of the present invention, processes of producing recombinant Herpesvirus vectors with low cytopathicity are provided, comprising the steps of c~ rin~ m~mm~ n cells with a first recombinant Herpesvirus vector c~ a G protein linked receptor gene and a second recombinant Herpesvirus vector 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 the recombinant virus by detecting G protein linked receptor c;~ ssion. ~urther, the G
WO 97/13866 PCT~US96/16368 protein linked receptor gene can be inserted into the TK locus. Within certain embodiments, 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 recombinant virus is deficient in the expression of one or more of the following: the TK
5 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 recombinant ~erpesvirus vectors with an in vitro cytopathicity 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%.
Within yet other aspects, recombinant ~Ierpesvirus vectors are provided which are capable of ex~ sshlg a G protein linked receptor with a surface receptor ession generally of greater than about 10,000 receptors/cell; typically in the range of about 25,000-200,000 receptors/cell; preferably in the range of about 200,000 to about 400,000 receptors/cell; or more preferably, greater than about 400,000 1 5 receptors/cell.
Yet other aspects of the present invention provide methods of using an ~x~l~s~ion ç~cett~ or gene delivery construct (e.g, a recombinant Herpesvirus vector) in the m~mlf~cture of a me~ ment for the ~ t of neuronal disorders.
These and other aspects of the present invention will become evident upon reference to the following detailed description and ~tt~rh~i drawings. In addition, various references are set forth which describe in more detail certain procedures and/or compositions, and are hereby incorporated by reference in their entirety as if each were specifically incorporated by reference.
Description of Figures Figure la is a sch~m~tic illustration of vhsA.
Figure lb is a sçh~ tic illustration of vTKhml-1.
Figure lc is a schematic illustration of vTKhml-2.
Figure ld is a schematic illustration of vTKhml-3.
Figure 2 is a srht~ tic diagram illustrating the detection of mlACHR 5' mRNA using a ribonuclease protection assay.
Figure 3 is a photograph of a urea~polyacrylamide gel which shows labeled probe that had hybridized to cellular RNA was subsequently identified following electrophoresis on an 8M urea~polyacrylamide gel and vi~ ii7ecl by autoradiography. A protected RNA fr~gment of 265 nt which corresponded to transcription of the insert from the CMV promoter was ~ietectç~i as early as 3 hours post W O 97/13866 PCTrUS96/16368 infection ("hpi"), reached high levels by 8 hpi, and m~;nt~ined high levels until 18 hpi.
(See Exarnple 2.) Figure 4 is a graph which shows saturation curves ~ple~ g the number of mlAchR expressed per Vero cell in samples harvested 2 to 36 hours post-S infection (hpi) in samples infected with one of the following: vTKhïml-1, vTKhml-2, and vTKhml-3.
Figure 5 is a graph which shows saturation curves ~ s~l-Lillg the number of mlAchR expressed in kansfected 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 vTKhrnl -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 representing a 15 comparison of receptor binding of vhsA to vTKhml-l .
Figure 8 is a photograph of a southern blot of viral DNA, cO~ g vhsA and vTKhml-1.
~ igure 9 is a photograph 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 saline and fixed with 3.2% formaldehyde for 10 min at room temperature. Cells were rinsed and permeabilized with 0.3% Triton X-100 for 3 min at room te~ c.aLule. Cells were then rinsed and incubated in primary antibody for 1 h, rinsed three times with saline, and incubated with fluorescentantibodies for 1 h at room ~ al~lre. Following this inc~lh~ticn, cells were rinsed, 25 mounted on a glass slide and viewed using an epifluorescence microscope with barrier filters to distinguish green from red fluorescence. The green signal is derived from fluorescein-isothiocyanate conjugated goat anti-rabbit antibody non-covalently attached to the ~ aly rabbit polyclonal antiserum anti-enolase. The orange signal is derived from tetramethyl rhodamine isothiocyanate conjugated goat anti-mouse antibody 30 ~tt~c~h~cl non-covalently to a mouse monoclonal antibody directed against the herpes protein ICPO.
Figure 10 is a photograph of a gel which shows protein synthesis in infected cells demonstrating that vTKhml-2, which is the backbone vector for vTKhm1-3 and vTKhml-1, does not alter protein synthesis after infection. Monolayers 35 of Vero cells were infected with virus for 1 h at 38~C, and rinsed with growth medium.
Cells were then incubated with growth medium lacking cold methionine. After 30 min, WO 97/13866 PCT~IIS96/16368 100 mCi/ml [35S] methionine was added for the ren7Ain(~er of the experiment. Cells monolayers were harvested in detergent buffers and proteins were identified on SDS
gels.
Figure 11 is a photograph of a DNA replication assay confirming the 5 phenotype of each strain of virus. Briefly, the results of this assay show that vTKhml-2 and vTKhm1-3 do not replicate in normal Vero cells, but do replicate in E5 cells, which express ICP4 and complement the defect in the virus.
Figures 1 2A and 1 2B are two photographs of sections of rat brains which have been infected with a recombinant Herpesvirus.
Figure 13 is a schematic illustration of the LAT promoters within an HSV-l transcript. Briefly, UL, US, TRL,TRS,IRL and IRS, represent HSV-l genomic sequences corresponding to unique-long, unique-short, t~rmin:~l repeat-long, terminal repeat-short, inverted repeat-long and inverted repeat-short, respectively. Lines with arrowheads denote HSV-l RNA transcripts. LAT promoters are ~ie~ign~te~l as LAPl 15 and LAP2. All cis transcription regulatory elements represented on the LAP1 promoter region from ~2 to -259 (1 lg,542 to 118,801 according to McGeoch et al., J. Gen. Virol.
69:1531-1574, 1988).
Figures 14A and 14B are graphs which show the schematic construction and relative activity in neural and non-neural cells of chimeric t;~ c;s~ion ç:l~settes 20 cont~inin~ LAP 1 promoters combined with NRSE silencer elem~nt~
Figures 15A and 15B are graphs which show the s~ h~m~tic construction and relative activity in neural and non-neural cells of several different chimeric expression cassettes co..l~ g a LAPl promoter and hCMV enhancer.
Figures 16A and 16B are graphs which show the schematic construction 25 and relative activity in neural and non-neural cells of several different chimeric e~res~ion ç~settes cont~ining LAPl promoters combined with both NRSE silencer elements and the hCMV enh~nrer.
Figures 1 7A and 1 7B are graphs which show the schematic construction and relative activity in neural and non-neural cells of several different chimeric 30 expression cassettes co..1~ p chimeric LAPl promoters in dirr~ te~l PC12 cells.
Detailed Description of the Invention ~ Prior to setting forth the invention, it may be helpful to an underst~n~ing 35 thereof to first set forth definitions of certain terms that will be used hereinafter.
W O 97/13866 PCT~US96/16368 A "neuronal-specific silencer element" refers to a cis-acting sequer~ce which down regulates or decreases the expression of a gene or sequence of interest in neuronal cells.
A "promoter element" refers to a sequence or sequences which facilitate 5 the initiation of transcription.
An "enhancer" refers to a cis-acting sequence which functions to increase transcription of a promoter.
As noted above, the present invention provides ~ s~ion c~settçs 10 which are suitable for a variety of in vivo and in vifro applications. As utilized herein, it should be understood that '~ t;ssion c~e1t~c" as utilized herein, refers to those vectors or molecules which are capable of directing the ~x~ sion of a sequence of interest in neuronal cells. Within one embodiment, such c~ssettes generally comprise one or more neuronal specific silencer elements, a promoter element operably linked to 15 a sequence of interest, and an enh~n~ er, wherein the enhancer and silencer elements are positioned such that they are not adJacent or next to one another.
Use of such ~res~ion c~Csettes provides the surprising and unexpected advantage of sl-hst~nti~lly increased expression in neuronal cells (such as PC12), as compared to non-neuronal cells (such as ~ero cells). Within certain embodiments the 20 vectors provided herein provide greater than about 3-fold, about 5-fold, about 8-fold, or about 10-fold levels of ~ ssion in neuronal cells as compared to non-neuronal cells.
Such expression cS~settes may be readily constructed given the disclosure provided herein, as well as standard recombinant techniques (see, e.g, Sambrook et al., "Molecular Cloning: A laboratory m~nu~l," 2nd ed., Cold Spring 25 Harbor Laboratory Press, Cold Spring Harbor, New York). Briefly, a wide variety of neuronal specific silencer elements may be utilized within the context of the present invention, including for example neural restrictive silencer elements "NRSE" (see Mori et al., Neuron 9(1):45-54, 1992; see also Schoenherr and Anderson, Science 267(5202):1360-1363, 1995, Devereux etal., Nucleic Acids Res. 12:387, 1984, and 30 Altschul et al., J. Mol. Biol. 215:403, 1990, which describe related consensus sequences); silencer elements which are specific for cholinergic cells (e.g., Baskin et al., Mol. Brain Res. 30(1):106-114, 1995; Li etal., J. Neurochem. 61(2):748-751, 1993);
silencer elements ofthe synapsin gene (Li etal., PN~S US~ 90(4):1460-1464, 1993), silencer elements of the do~ hle beta-hydroxylase gene (Ishiguro et al., J. Biol.
35 Chem. 268(24):17g84-17987, 1993); silencers of neuronal Na, K-ATPase subunit genes CA 02234604 l998-04-l4 WO 97/13866 PC~US~6~6368 (Pathak et al., Nucleic Acids ~es. 22:4748, 1994) and silencers of type Il sodium channel genes (Kraner et al., Neuron 9:37-44, 1992).
The above-noted ~ ,rcssion c~c.cett~ likewise contain promoter elements which facilitate the initiation of transcription. A wide variety of promoters 5 may be utilized within the context of the present invention, including for example, both viral and cellular promoters. R~lcsell~Live exarnples of viral promoters includeMoMLV LTR, RSV LTR, adenoviral promoter (Ohno etal., Science 265:781-784, 1994), late parvovirus promoter (Koering et al., Hum. Gene Therap. 5:457-463, 1994);
Herpes promoters (see McGeoch et al., J. Gen. Yirol. 69:1531-1574, 1988 and Ward10 and Roizman, 7:rends in Gen. 10(8):267-274, 1994) such as the TK promoter, ICP6 promoter, ICP4 promoter, VP16 promoter or latency-associated promoters (e.g., HSV-I
LAT promoter (Ho and Mocarski, Proc. Natl Acad. Sci. USA 86:7596-7600, 1989)), SV40 promoters (Subramani etal., MoL Cell. Biol. 1:854-864, 1981), cytomegalovirus immediate early promoter (Boshart et al., Cell 41:521-530, 1985), and the 15 cytomegalovirus imm~ te late promoter. Representative examples of cellular promoters include the neomycin phosphotransferase promoter; metallothionein-l promoter (Palmiter et al., U.S. Patent No. 4,579,821, and the mouse VH promoter (Loh etal., Cell 33:85-93, 1983). Otner suitable promoters include tissue or cell-specific promoters (see e.g, WO 91/02805; EP 0,415,731; and WO 90/07936). Representative 20 examples of suitable neuron specific promoters include tocl tubulin (Gloster etal., J. Neurosci 14(12):7319-30, 1994), and the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene promoter (Milton et al., J. Biol. Chem. 270(25):15143-7, 1995).
The above-noted t;x~l~;ssion ç~ett~ may also contain enhancer elements, which are cis-acting sequences which function to upregulate transcription 25 from a nearby promoter. Representative examples include the CMV enh~ncer, theSV40 enhancer, and the 5' enh~n~er of tne MMTV LTR (Mellentin-Michelotti et al., J.
Biol. Chem. 269(50):31983-90, 1994) The above-noted ~x~ s~ion c~settes may be readily constructed in order to allow subst~nt~ y increased levels of expression in neuronal cells. For30 example, within one embodiment expression c~cse~tes are constructed with the following ordered elements (in a 5' to 3' orientation): the LAP1 promoter operably ~ linked to a sequence of interest, followed by the hCMV enhancer. Within other embo-1iment~ ~x~i~sion cassettes are provided with the following ordered elements:
one or more neuronal restrictive silencer elements, a LAP 1 promoter operably linked to 35 a sequence of interest, and an hCMV enhancer. Within certain preferred embodiments, the neuronal restrictive silencer elements may be in the same transcriptional orientation W O 97/13866 PCTrUS96/16368 as the LAPI promoter, or ~It~ tively, in an opposite and divergent transcriptional orientation.
Such c~ c~ion c~settes may be utilized in a variety of constructs and methods, as described in more detail below. For example, such expression cassettes S may be delivered directly to an in vitro cell culture or to a warrn-blooded animal by a variety of techniques, including for exarnple by itself (WO 90/11092), in liposomes, condensed with polycations linked or unlinked to killed adenovirus (Curiel et al., Hurn Genc ~her. 3:147-154, 1992), or linked to a ligand (Wu etal., J. Biol. Chem 264: 16985- 16987, 198g).
GENE DELIVERY CONSTRUCTS
In addition to expression c~cettes, which are useful by themselves for transfecting cells or therapeutic purposes, the present invention also provides gene delivery constructs which are useful for carrying and/or delivering the above-described 15 ~ ession cassettes. Rc~lcscnL~live exarnples of such constructs include a variety of non-viral and viral vectors, as described below, as well as cells which are capable of producing such vectors.
For example, within one aspect of the invention retroviral vectors may be utilized as gene delivery constructs suitable for delivering the above-noted expression 20 constructs. Represelllative examples of such retroviral vectors include those described within EP 0,415,731, WO 90/07936, WO 91/0285, WO 94/03622, WO 93/25698, WO
93/25234, U.S. Patent No. 5,219,740, WO 93/11230, WO 93/10218, U.S. Patent No. 4,777,127, EP 0,345,242 and WO 91/02805).
Other exarnples of suitable gene delivery constructs may be found in the 25 Herpesvirus farnily. Briefly, suitable members of the Herpesviridae include both primate Herpesviruses, and nonprimate Herpesviruses such as avian Herpesviruses.Representative examples of suitable Herpesviruses include Herpes Simplex Virus Type 1 (McKnight etal., Nuc. Acids Res. 8:5949-5964, 1980; Fields etal., Fundamental Virolo~y, Raven Press, N.Y. ~ 1986)), Herpes Simplex Virus Type 2 (Swain and 30 Galloway, J. Virol. 46:1045-1050, 1983), Varicella Zoster Virus (Davison and Scott, J.
Gen. Yirol. 67:1759-1816, 1986) and Epstein-Barr virus (Baer et al., Nature (London) 310:207-311, 1984). Particularly preferred Herpesvirus vectors include those described below, which are deficient in the c2~lc~ion of one or more of: the virion host shut-off protein VHS; a replication loci such as IC~P~; or thymidine kinase. Representative 35 examples of such vectors include vTKhml-l, vTKhml-2 and vTKhrnl-3.
CA 02234604 l998-04-l4 W O 97/13866 PC~nUS96~I6368 Herpesviruses may be readily obtained from commercial sources such as the American Type Culture Collection ("ATCC", Rockville, Maryland). Deposits of certain of the above-identified Herpesviruses may be readily obtained fronn the ATCC, for example: ATCC No. VR-539 (Herpes simplex type l); ATCC Nos. VR-734 and S VR-540 (Herpes Simplex type 2); and ATCC No. VR-586 (Varicella Zoster Virus).
Herpesviruses may also be readily isolated and identified from naturally occurring sources (e.g., from an infected animal).
In addition to retroviral vectors and Herpes viral vectors, a wide variety of other gene delivery constructs may be utilized to deliver ~;x~lc;ssion cassettes, including for example constructs derived from adenovirus ~Rosenfeld etal., Science 252:431-434, 1991; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS
90(24):11498-502, 1993; Levrero etal., Gene 101(2):195-202, l991), and Guzman et al., Circulation 88(6):2838-48, 1993; alphaviruses such as Semliki Forest Virus and Sindbis Virus (Xiong et al., Science 243:1188, 1989; Raju and Huang, J. ~ir.
65(5):2501-2510, 1991; Hertz and Huang, J. Vir. 66(2):857-864, 1992, WO 92/10578;
WO 95/07994; U.S. Patent No. 5,091,309); influenza virus (Luytjes et al., Cell 59: 1107-1113, 1989; McMicheal et al., N. Eng J. Med. 309:13-17, 1983; and Yap et al., Nature 273:238-239, 1978); pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch etal., PNAS 86:317-321, 1989; Flexner etal., Ann. N.Y. Acad. Sci. 569:86-103, 1989; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114, 1979); parvovirus such as adeno-associated virus ~S~mnl~ki etal., J. Yir. 63:3822-3828, 1989; Plotte etal., G. Biol. Chem. 268:3781-3790, 1993; Flotte etal., PNAS 90(22):10613-10617, 1993; WO95/13365); and HI~I
(Poznansky, J. Virol. 65:532-536, 1991).
Other non viral vector systems that may also be utilized include a variety of nucleic acid based l~ s~ ion systems (e.g, based on T7 or SP6 promoters, see generally, WO 95/07994). Such vector systems may be ~-1mini~tered and prepared as described above (e.g, in liposomes, condensed with polycations, or linked to a ligand).
~EOUENCES OF INTEREST
A wide variety of heterologous nucleic acid sequences (also referred to as nucleic acid segrnent~ or molecules) may be expressed by the ~x~ ion cassettes and gene delivery constructs of the present invention, including for example, ~nti~çn~e~
CA 02234604 l998-04-l4 W O 97tl3866 PCT~US96/16368 ribozyme or regulatory sequences, as well as a wide variety of proteins such as immunogenic portions of disease-associated antigens, immunologically active molecules, replacement genes and toxic genes.
Representative examples of antisense and ribozyme sequences include 5 those sequences which inhibit, for exatnple, tumor cell growth, viral replication, or a genetic disease by preventing the cellular synthesis of critical proteins. Examples of such ~nti~nce sequences include antisense ABL (Fainstein etal., Oncogene 4:1477-1481, 1989), zinti~çn~e HER2 (Coussens et al., Science 230:1132-1139, 1985), ~nticçll~e myc (Stanton et al., Nature 310:423~25, 1984), ~nti~n~e ras and antisense CPP32 or 10 ice proteases.
Representative examples of ribozytne sequences include h~mmerhes~l ribozymes (for example, as described by Forster and Symons, Cell 48:211-220, 1987;
Haseloff and Gerlach, Nature 328:596-600, 1988, Walbot and Bruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature 334:585, 1988) and hairpin ribozymes15 (for example, as described by Haseloffetal., U.S. Patent No. 5,254,678 and Hempel et al., European Patent Publication No. 0 360 257) which have the ability to specifically target, cleave and inactivate RNA or mRNA. Briefly, the sequence requirement for the hairpin ribozyme is any RNA sequence consisting of NNNBN*GUCNNNN~ (where N*G is the cleavage site, wherein B is any of G, 20 C, or U, and where N is any of G, U, C, or A) (Sequence I.O. No. 1). The sequence requirement at the cleavage site for the h~mmerhe~-l ribozyme is any RNA sequence consisting of NUX (where N is any of G, U, C, or A and X represents C, U or A) can be targeted. Accordingly, the same target within the hairpin leader sequence, GUC, is useful for the hammerhead ribozyme. The additional nucleotides of the hammerhead25 ribozyme or hairpin ribozyme is c~et~rmined by the target fl~nkinp nucleotides and the hammerhead c~ n~en~us sequence (see Ruffner et al., Biochemistry 29:10695-10702,1990).
"Disease-associated" antigens should also be understood to include all, or various portions (e.g, immunogenic portions) of eukatyotic (including for example, 30 parasites), prokaIyotic (e.g, bacterial) or viral pathogens. Other "disease-associated"
antigens include tumor-associated antigens such as ras, p53 and CEA.
Immunologically active molecules may also be ~ essed by the expression c~ettes and gene delivery constructs described herein. As utilized within the context of the present invention, it should be understood that "immunologically 35 active molecules" refers to those molecules which can either increase or decrease the recognition, presentation or activation of a cell-mediated or humoral immune response.
CA 02234604 l998-04-l4 W O 97/13866 PCT~US96~636~
Representative examples of immunologically active molecules include Iymphokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, ~L-8, IL-9, IL-10, IL-11, IL-12 (WO
90/05147; EPO 433,827), IL-13 (WO g4/04680), IL-14, IL-15, a, 13,or ~ r~
GM-CSF, M-CSF-l, G-CSF, ICAM-1 (Simmons etal., Nature 331:624-627, 1988), 5 ICAM-2 (Singer, Science 255: 1671, 1992), 13-microglobulin (Parnes et al., PNAS
78:2253-2257, 1981), HLA Class I, HLA Class II molecules, B7 (Freeman etal., J.
Immun. 143:2714, 1989), and B7-2, as well as their respective receptors. Other biologically active molecules that may likewise be utilized in the context of the present invention include neurotrophins such as nerve growth factor (N~F), brain derived10 growth neurotrophic factor (BDGF), glial-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), tumor necrosis factor (TNF) as well as their respective receptors.
Within certain aspects of the present invention, the ~ e~sion c~etteS
and gene delivery constructs described herein may direct the expression of more than 15 one heterologous sequence. Such multiple se~uences may be controlled either by a single promoter, or ~ltern~tively~ by additional secondary promoters (e.g, Tnt~rn~l Ribosome Entry Sites or "IRES"). Within further embodiments of the invention, expression cassettes or gene delivery constructs are provided which direct the expression of heterologous sequences which act synergistically (e.g., a disease-20 associated antigen, and an immlml logically active molecule, such as IL-2, IL-12 or r-interferon).
Within other aspects of the invention, the t;~ t;ssion c~ettes or gene delivery constructs described herein may direct the expression of one or more heterologous sequences which encode "replacement" genes. As utilized within the 25 context of the present invention, the term "replacement genes" refers to a nucleic acid molecule which encodes a therapeutic protein that is capable of preventing, inhibiting, stabilizing or l~:velsillg an inherited or ncninherited genetic defect. Representative exarnples of such genetic defects include disorders in metabolism, immnne regulation, horrnonal regulation, and enzymatic or membrane associated structural function.
30 Specific examples include ~17hPimer's Disease (see, for example, Goat et al., Nature 349:704, lg91; Sherringt-n etal., Nature 375:754, 1995; Levy-Labad etal., Science 269:973, 1995) and Huntington's Disease (see EP 0 614,977 and WO 94/21790).
Replacement genes may also be ~1mini~tered for specific conditions, such as the ~(1mini~tration of anti-apoptotic genes or sequences such as Bc1-2, BclX or Bax (Oitvai 35 etal., Cell 74:60g-619, 1993), neuronal apoptosis inhibitory protein ("NAIP", Roy W O 97/13866 PCTrUS96/16368 et al., Cell 80:1-20, 1995), or TNF (Zheng et al., Nature 377:348-351, 19g5) in order to inhibit or remedy conditions or disease where apoptosis occurs.
Representative exarnples of toxic genes which may be expressed and/or delivered by the e~Le~ion csl~sett~s and gene delivery constructs provided herein include genes which encode proteins such as abrin (Wood etal., Eur. J. Biochem.
198:723-732, 1991), diphtheria toxin (Tweten et al., J: Biol. C~em. 260: 10392- 10394, 1985), antiviral protein (Barbieri et al., Biochem. J: 203:55-59, 1982; Irvin et al., Arch.
Biochem. & Biophys. 200:418-425, 1980), cholera toxin (~ek~l~nos etal., Nature 306:551-557, 1983; Sanchez and Holmgren, PNAS ~6:481-485, 1989), gelonin (Stirpeet al., J. Biol. C~em. 255:6947-6953, 1980), pokeweed (Irvin, Pharmac. Ther. 21 :371 -387, 1983), ricin (Larnb etal., E~ur. J. Biochem. 148:265-270, 1985), Shigella toxin (Calderwood et al., PN~lS 84:4364-4368, 1987), tritin, and Pseudomonas exotoxin A
(Carroll and Collier, J. Biol. Chem. 262:8707-8711, 1987).
Within other aspects of the invention, heterologous sequences should be understood to include gene products which activate a non-toxic product into a toxic product. Representative examples of such gene products include thymidine kinaseswhich activate a nucleoside analogue such as acyclovir or gancyclovir, as well as other "prodrugs" (see WO 93/10218, WO 93/01281; WO 93/08843; WO 93/08844; and WO
so/07936 ) As should be evident from the above discussion, expression cassettes of the present invention may be utilized to express a wide variety of additional sequences, including for exarnple those encoding receptors (including, for exarnple, G protein linl~ed receptors as described in more detail below), regulatory proteins, enzymes and structural proteins not specifically set forth above.
Sequences which encode the a~ove-described heterologous genes may be readily obtained from a variety of sources. For exarnple, plasmids which containsequences that encode imm~ln-~logically active molecules may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford, F.n~l~n~l). Representative sources sequences which encode the above-noted imrnune accessory molecules include ATCC No. 20663 (which contains sequences encoding alpha hlLelrel~on), ATCC Nos. 31902 and 39517 (which containssequences encoding beta hlLelr~lol1), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (which contains sequences encoding Interleukin-2), ATCC Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), ATCC No. 57592 (which contains sequences encoding Interleukin-4), ATCC Nos. 59394 and 59395 WO 97tl3866 PC~fUSg6~636~
(which contain sequences encoding Interleukin-5), and ATC(~ No. 67153 (which contains sequences encoding Interleukin-6). It will be evident to one of skill in the art that one may utilize either the entire sequence of the protein, or an a~lo~l;ate portion thereof which encodes the biologically active portion of the protein.
S Alternatively, known cDNA sequences which encode cytotoxic genes or other heterologous genes may be obtained from cells which express or contain such sequences. Briefly, within one embodiment mRNA from a cell which expresses the gene of interest is reverse transcribed with reverse trans.~ se using oligo dT or random primers. The single stranded cDNA may then be amplified by PCR (see U.S.
Patent Nos. 4,683,2~2, 4,683,195 and 4,800,159. See also PCR Technology: Principles and Applications ~or DNA Amplification, Erlich (ed.), Stockton Press, 1989 all of which are incorporated by reference herein in their entirety) lltili~ing oligonucleotide primers complementary to sequences on either side of desired sequences. In particular, a double stranded DNA is denatured by heating in the presence of heat stable Taqpolymerase, sequence specific DNA primers, ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA.
Sequences which encode the above-described genes may also be synth~i7~-1, for example, on an Applied Biosystems Inc. DNA synth~?si7~r (e.g., ABI
Technical Field The subject invention is generally directed to a recombinant Herpesvirus vectors and more specifically, recombinant Herpesvirus vectors with a high rate of expression of foreign gene sequences and/or a low cytopathicity in neuronal cells.
Back~round of the Invention The capacity to introduce genetic sequences into a m7~mm~ n cell and to 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 de~igning a therapeutic basis for the treatment of disease.
The introduction of a genetic sequence into a m~mm~ n host cell may be facilitated by first introducing the sequence into a suitable vector. However, vectors suitable for use in nonmitotic cells, such as neural or neuronal cells, has proven challenging. In addition, whereas most tissues in the body are readily zl~cf ~ihle 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-replicative state of most nerve cells, present peculiar challenges when ~le~ ning gene therapy systems.
These challenges have hindered the possible tre~tmenf of neurological disorders such as brain tumors, de~ne.dli~e disorders (multiple sclerosis, Parkinson's disorder, Alzheimer's disease, ~ y~Llophic lateral sclerosis), disorders caused by abnormal expression of genes, inherited disorders caused by a known gene defect (HPRT in Lesch-Nyhan disorder; retinblastoma ~Lee et al., Sci. 235:1394, 1987);
glucocerebrosidase (Sorge et al., Proc. Natl. Acad. Sci. USA 84:906, 1987); and Duchenne's muscular dy~lloplly (Monaco et al., Nature 321:443, 1986)) and acute injuries to the brain or peripheral nervous tissue, for exarnple from a stroke, brain injury, or spina} cord injury.
Although many viral vector systems have been developed, there has been difficulty adapting these systems for neuronal cells. For example, although retroviral vectors have been used to L~ l genes into neuronal cells in vitro (Price et al., Proc.
Natl. Acad. Sci USA 34:156-160, 1987), and in vivo ~Culver etal., Science 256:1550, 1992); Price et al., supra~, they have not proven useful in delivering genes to a large plupollion of cells in the nervous system. Other viral vector systems also have characteristics limiting their usefulness for gene transfer into neuronal cells, such as:
5 rapidly clearing Iytic infections (e.g, adenovirus~ vaccinia virus), small genome size (SV40, polyoma), or limited cell tropism (EBV, bovine papilloma virus).
A Herpes Simplex Virus-l (HSV-l) vector has been shown to be useful for infecting a wide variety of cells, including neuronal cells (Spear and Roizrnan, DN~
Tz~n20~ Viruses, Cold Spring Harbor Laboratory, NY, pp. 615-746). Briefly, HSV-I can 10 exist in a latent state in neural cells (Stevens, Microbiol. Rev. 53:318, 1989) allowing for m~inten~nce of the vector. Additionally, the viral genome of HSV-l is very large (150 kb) and may accommodate large nucleic acid segment~.
Geller et al. (PCT WO 90/09441) developed a HSV-l virus-based vector, which, while offering advantages over plasmid-based vectors, has failed to be 15 efficacious in several in~t~nres. These vectors suffer from low gene ~x~le3~ion and high cytopathicity, thus severely limi~ing their use in gene transfer. While others have tried to increase expression by using a variety of promoters (Tackney, et al, ~ Virol.
52:606, 1984), cytopathicity has been shown to be a persistent problem, even in those viral vectors which are replication deficient (Johnson etal., J. I~irol. 66:2952, 1992;
20 Johnson etal., Mol. Brain Res. 12:95, 1992). For long-term expression in neuronal cells, it is necessary to have a viral vector that demon~ll~es low cytopathicity.
In view of the inability of current HSV-I vectors to adequately account for the balance of cytopathicity and gene ~ ession, it is apparent that there exists a need for new and additional methods and compositions which address and rectify the problem.
25 The present invention fulfills this need, and further provides related advantages.
Summarv of the Invention Briefly stated, the present invention provides t;~ression ç~csett~s capable of e2~ ssillg a sequence of interest. Within one aspect such expression 30 c~sett.?s comprise one or more neuronal specific silencer elements, a promoter element operably linked to a sequence of interest, and an enhancer, wherein the enhancer and silencer elements are positioned such that they are not adjacent to one another.Within one embodiment, the silencer element is a neuronal restrictive silencer element, and the promoter element is selected from the group consisting of 35 CMV, SV40, herpes promoters and adenovirus promoters.
W O 97113866 PCT~US96/16368 Within other aspects, expression cassettes are provided a LAT promoter operably linked to a sequence of interest, followed by an enhancer. Within further embofiiment~, such e~p~ ion cassettes further comprise a neuronal-specific silencer element, wherein the enhancer and silencer elements are positioned on the vector such S that they are not adjacent to one another. Within further embotliment~, the expression cassettes are capable of expressing a sequence selected from the group consisting of antisense and ribo~yme sequences, sequences which encode disease-associated antigens, sequences which encode imrnunologically active molecules, replacement genes, arld toxic genes.
Within other aspects of the present invention, gene delivery construct, as well as host cells are provided which contain one of the above-identified expression ç~c~ett~s. Representative examples of suitable host cells include cells from a warm-blooded animal or vertebrate, and includes in particular neuronal cells such as cortical neurons, cerebellar granule cells, retinal ganglia cells, hippocampal neurons, peripheral 15 sensory neurons, and motor neurons.
Within another aspect of the present invention, methods are provided for producing a protein, comprising the steps of (a) introducing an e~res~ion cassette or ~ene delivery construct as described herein into a host cell, and (b) cultllring the host cell under conditions, and for a time sufficient, to permit ~ ion of the protein.
20 Within further embo-liment~, such methods further comprise the step of purifying the protein.
Within other aspects of the present invention, methods are provided for introducing a selected se~luence of interest into neuronal cells of a warm-blooded animal, comprising the step of ~lmini~tering to said animal an expression ~ ettP or 25 gene delivery construct provided herein. Such vectors may be ~flmini~tered by a variety of routes, including for example, subcutaneously, intracranially, intradermally,intr~mll~c~ rly, hllla~ oneally, or intravenously. Within other embot1imt~.nt~, the vector may be directly ~imini~tered to a tumor.
Within other aspects, methods are provided for introducing a selected 30 sequence of interest into an in vitro culture cont~ining neuronal cells, compri~ing the step of introducing an expression cassette or gene delivery construct as described herein ~ into an in vitro culture cont~ining neuronal cells.
The present invention also provides recombinant Herpesvirus vectors capable of directing expression of a C~ protein linked receptor gene. Within certain 35 embo~liment~ of the invention, the recombinant viruses direct the expression of such CA 02234604 l998-04-l4 W O 97/13866 PCTrUS96/16368 genes in non-mitotic m~mm~ n cells, and more preferably, in m~mmz~ n neuronal cells.
Within other aspects of the present invention, recombinant Herpesvirus vectors are provided which are capable of directing the c;x~es~ion of an antisense 5 transcript of the G protein linked receptor gene.
In one embodiment of the invention, recombinant Herpesvirus vectors 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 embodiment of the present invention, the gene encoding a G
protein linked receptor or a antisense segm~nt thereof is inserted in the TK locus of a Herpesvirus genome. For example, the antisense se~ment may be a ~-HT2 receptor gene. Numerous G-protein linked receptor genes may be utilized within the context of the present invention, including, by way of example, a human Ml mn~c~rinic acetylcholine receptor gene or an adrenergic receptor.
Within other aspects of the invention, methods of treating m:~mm~l~ for neurological disorders are provided, com~ri~in~ the step of ~mini~tering to a m~mm~l a composition comprising an expression cassette or gene delivery construct such as a recombinant Herpesvirus vector, as described above. Within certain embodiments, this may be accomplished in combination with a phs~rm~ce~ltically acceptable carrier or diluent.
Within certain embo-lim~nt.c, the ~-1mini~tration of pharmaceutical compositions may be accomplished by, for example, by stereotactically microinjection, a time release mech~ni.cm, a sustained release merh:~ni.~m, chronic infusion, or ex vivo m~rnm~ n cells infected with or c~r~ a recombinant Herpesvirus vector~ gene delivery constructs, or expression cassette according to the present invention.
Another aspect of the present invention provides ph~rmzlrelltical compositions comprising an expression cassette or gene delivery construct of thepresent invention and a ph~rm~- eutically acceptable carrier or diluent.
Within yet other aspects of the present invention, processes of producing recombinant Herpesvirus vectors with low cytopathicity are provided, comprising the steps of c~ rin~ m~mm~ n cells with a first recombinant Herpesvirus vector c~ a G protein linked receptor gene and a second recombinant Herpesvirus vector 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 the recombinant virus by detecting G protein linked receptor c;~ ssion. ~urther, the G
WO 97/13866 PCT~US96/16368 protein linked receptor gene can be inserted into the TK locus. Within certain embodiments, 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 recombinant virus is deficient in the expression of one or more of the following: the TK
5 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 recombinant ~erpesvirus vectors with an in vitro cytopathicity 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%.
Within yet other aspects, recombinant ~Ierpesvirus vectors are provided which are capable of ex~ sshlg a G protein linked receptor with a surface receptor ession generally of greater than about 10,000 receptors/cell; typically in the range of about 25,000-200,000 receptors/cell; preferably in the range of about 200,000 to about 400,000 receptors/cell; or more preferably, greater than about 400,000 1 5 receptors/cell.
Yet other aspects of the present invention provide methods of using an ~x~l~s~ion ç~cett~ or gene delivery construct (e.g, a recombinant Herpesvirus vector) in the m~mlf~cture of a me~ ment for the ~ t of neuronal disorders.
These and other aspects of the present invention will become evident upon reference to the following detailed description and ~tt~rh~i drawings. In addition, various references are set forth which describe in more detail certain procedures and/or compositions, and are hereby incorporated by reference in their entirety as if each were specifically incorporated by reference.
Description of Figures Figure la is a sch~m~tic illustration of vhsA.
Figure lb is a sçh~ tic illustration of vTKhml-1.
Figure lc is a schematic illustration of vTKhml-2.
Figure ld is a schematic illustration of vTKhml-3.
Figure 2 is a srht~ tic diagram illustrating the detection of mlACHR 5' mRNA using a ribonuclease protection assay.
Figure 3 is a photograph of a urea~polyacrylamide gel which shows labeled probe that had hybridized to cellular RNA was subsequently identified following electrophoresis on an 8M urea~polyacrylamide gel and vi~ ii7ecl by autoradiography. A protected RNA fr~gment of 265 nt which corresponded to transcription of the insert from the CMV promoter was ~ietectç~i as early as 3 hours post W O 97/13866 PCTrUS96/16368 infection ("hpi"), reached high levels by 8 hpi, and m~;nt~ined high levels until 18 hpi.
(See Exarnple 2.) Figure 4 is a graph which shows saturation curves ~ple~ g the number of mlAchR expressed per Vero cell in samples harvested 2 to 36 hours post-S infection (hpi) in samples infected with one of the following: vTKhïml-1, vTKhml-2, and vTKhml-3.
Figure 5 is a graph which shows saturation curves ~ s~l-Lillg the number of mlAchR expressed in kansfected 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 vTKhrnl -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 representing a 15 comparison of receptor binding of vhsA to vTKhml-l .
Figure 8 is a photograph of a southern blot of viral DNA, cO~ g vhsA and vTKhml-1.
~ igure 9 is a photograph 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 saline and fixed with 3.2% formaldehyde for 10 min at room temperature. Cells were rinsed and permeabilized with 0.3% Triton X-100 for 3 min at room te~ c.aLule. Cells were then rinsed and incubated in primary antibody for 1 h, rinsed three times with saline, and incubated with fluorescentantibodies for 1 h at room ~ al~lre. Following this inc~lh~ticn, cells were rinsed, 25 mounted on a glass slide and viewed using an epifluorescence microscope with barrier filters to distinguish green from red fluorescence. The green signal is derived from fluorescein-isothiocyanate conjugated goat anti-rabbit antibody non-covalently attached to the ~ aly rabbit polyclonal antiserum anti-enolase. The orange signal is derived from tetramethyl rhodamine isothiocyanate conjugated goat anti-mouse antibody 30 ~tt~c~h~cl non-covalently to a mouse monoclonal antibody directed against the herpes protein ICPO.
Figure 10 is a photograph of a gel which shows protein synthesis in infected cells demonstrating that vTKhml-2, which is the backbone vector for vTKhm1-3 and vTKhml-1, does not alter protein synthesis after infection. Monolayers 35 of Vero cells were infected with virus for 1 h at 38~C, and rinsed with growth medium.
Cells were then incubated with growth medium lacking cold methionine. After 30 min, WO 97/13866 PCT~IIS96/16368 100 mCi/ml [35S] methionine was added for the ren7Ain(~er of the experiment. Cells monolayers were harvested in detergent buffers and proteins were identified on SDS
gels.
Figure 11 is a photograph of a DNA replication assay confirming the 5 phenotype of each strain of virus. Briefly, the results of this assay show that vTKhml-2 and vTKhm1-3 do not replicate in normal Vero cells, but do replicate in E5 cells, which express ICP4 and complement the defect in the virus.
Figures 1 2A and 1 2B are two photographs of sections of rat brains which have been infected with a recombinant Herpesvirus.
Figure 13 is a schematic illustration of the LAT promoters within an HSV-l transcript. Briefly, UL, US, TRL,TRS,IRL and IRS, represent HSV-l genomic sequences corresponding to unique-long, unique-short, t~rmin:~l repeat-long, terminal repeat-short, inverted repeat-long and inverted repeat-short, respectively. Lines with arrowheads denote HSV-l RNA transcripts. LAT promoters are ~ie~ign~te~l as LAPl 15 and LAP2. All cis transcription regulatory elements represented on the LAP1 promoter region from ~2 to -259 (1 lg,542 to 118,801 according to McGeoch et al., J. Gen. Virol.
69:1531-1574, 1988).
Figures 14A and 14B are graphs which show the schematic construction and relative activity in neural and non-neural cells of chimeric t;~ c;s~ion ç:l~settes 20 cont~inin~ LAP 1 promoters combined with NRSE silencer elem~nt~
Figures 15A and 15B are graphs which show the s~ h~m~tic construction and relative activity in neural and non-neural cells of several different chimeric expression cassettes co..l~ g a LAPl promoter and hCMV enhancer.
Figures 16A and 16B are graphs which show the schematic construction 25 and relative activity in neural and non-neural cells of several different chimeric e~res~ion ç~settes cont~ining LAPl promoters combined with both NRSE silencer elements and the hCMV enh~nrer.
Figures 1 7A and 1 7B are graphs which show the schematic construction and relative activity in neural and non-neural cells of several different chimeric 30 expression cassettes co..1~ p chimeric LAPl promoters in dirr~ te~l PC12 cells.
Detailed Description of the Invention ~ Prior to setting forth the invention, it may be helpful to an underst~n~ing 35 thereof to first set forth definitions of certain terms that will be used hereinafter.
W O 97/13866 PCT~US96/16368 A "neuronal-specific silencer element" refers to a cis-acting sequer~ce which down regulates or decreases the expression of a gene or sequence of interest in neuronal cells.
A "promoter element" refers to a sequence or sequences which facilitate 5 the initiation of transcription.
An "enhancer" refers to a cis-acting sequence which functions to increase transcription of a promoter.
As noted above, the present invention provides ~ s~ion c~settçs 10 which are suitable for a variety of in vivo and in vifro applications. As utilized herein, it should be understood that '~ t;ssion c~e1t~c" as utilized herein, refers to those vectors or molecules which are capable of directing the ~x~ sion of a sequence of interest in neuronal cells. Within one embodiment, such c~ssettes generally comprise one or more neuronal specific silencer elements, a promoter element operably linked to 15 a sequence of interest, and an enh~n~ er, wherein the enhancer and silencer elements are positioned such that they are not adJacent or next to one another.
Use of such ~res~ion c~Csettes provides the surprising and unexpected advantage of sl-hst~nti~lly increased expression in neuronal cells (such as PC12), as compared to non-neuronal cells (such as ~ero cells). Within certain embodiments the 20 vectors provided herein provide greater than about 3-fold, about 5-fold, about 8-fold, or about 10-fold levels of ~ ssion in neuronal cells as compared to non-neuronal cells.
Such expression cS~settes may be readily constructed given the disclosure provided herein, as well as standard recombinant techniques (see, e.g, Sambrook et al., "Molecular Cloning: A laboratory m~nu~l," 2nd ed., Cold Spring 25 Harbor Laboratory Press, Cold Spring Harbor, New York). Briefly, a wide variety of neuronal specific silencer elements may be utilized within the context of the present invention, including for example neural restrictive silencer elements "NRSE" (see Mori et al., Neuron 9(1):45-54, 1992; see also Schoenherr and Anderson, Science 267(5202):1360-1363, 1995, Devereux etal., Nucleic Acids Res. 12:387, 1984, and 30 Altschul et al., J. Mol. Biol. 215:403, 1990, which describe related consensus sequences); silencer elements which are specific for cholinergic cells (e.g., Baskin et al., Mol. Brain Res. 30(1):106-114, 1995; Li etal., J. Neurochem. 61(2):748-751, 1993);
silencer elements ofthe synapsin gene (Li etal., PN~S US~ 90(4):1460-1464, 1993), silencer elements of the do~ hle beta-hydroxylase gene (Ishiguro et al., J. Biol.
35 Chem. 268(24):17g84-17987, 1993); silencers of neuronal Na, K-ATPase subunit genes CA 02234604 l998-04-l4 WO 97/13866 PC~US~6~6368 (Pathak et al., Nucleic Acids ~es. 22:4748, 1994) and silencers of type Il sodium channel genes (Kraner et al., Neuron 9:37-44, 1992).
The above-noted ~ ,rcssion c~c.cett~ likewise contain promoter elements which facilitate the initiation of transcription. A wide variety of promoters 5 may be utilized within the context of the present invention, including for example, both viral and cellular promoters. R~lcsell~Live exarnples of viral promoters includeMoMLV LTR, RSV LTR, adenoviral promoter (Ohno etal., Science 265:781-784, 1994), late parvovirus promoter (Koering et al., Hum. Gene Therap. 5:457-463, 1994);
Herpes promoters (see McGeoch et al., J. Gen. Yirol. 69:1531-1574, 1988 and Ward10 and Roizman, 7:rends in Gen. 10(8):267-274, 1994) such as the TK promoter, ICP6 promoter, ICP4 promoter, VP16 promoter or latency-associated promoters (e.g., HSV-I
LAT promoter (Ho and Mocarski, Proc. Natl Acad. Sci. USA 86:7596-7600, 1989)), SV40 promoters (Subramani etal., MoL Cell. Biol. 1:854-864, 1981), cytomegalovirus immediate early promoter (Boshart et al., Cell 41:521-530, 1985), and the 15 cytomegalovirus imm~ te late promoter. Representative examples of cellular promoters include the neomycin phosphotransferase promoter; metallothionein-l promoter (Palmiter et al., U.S. Patent No. 4,579,821, and the mouse VH promoter (Loh etal., Cell 33:85-93, 1983). Otner suitable promoters include tissue or cell-specific promoters (see e.g, WO 91/02805; EP 0,415,731; and WO 90/07936). Representative 20 examples of suitable neuron specific promoters include tocl tubulin (Gloster etal., J. Neurosci 14(12):7319-30, 1994), and the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene promoter (Milton et al., J. Biol. Chem. 270(25):15143-7, 1995).
The above-noted t;x~l~;ssion ç~ett~ may also contain enhancer elements, which are cis-acting sequences which function to upregulate transcription 25 from a nearby promoter. Representative examples include the CMV enh~ncer, theSV40 enhancer, and the 5' enh~n~er of tne MMTV LTR (Mellentin-Michelotti et al., J.
Biol. Chem. 269(50):31983-90, 1994) The above-noted ~x~ s~ion c~settes may be readily constructed in order to allow subst~nt~ y increased levels of expression in neuronal cells. For30 example, within one embodiment expression c~cse~tes are constructed with the following ordered elements (in a 5' to 3' orientation): the LAP1 promoter operably ~ linked to a sequence of interest, followed by the hCMV enhancer. Within other embo-1iment~ ~x~i~sion cassettes are provided with the following ordered elements:
one or more neuronal restrictive silencer elements, a LAP 1 promoter operably linked to 35 a sequence of interest, and an hCMV enhancer. Within certain preferred embodiments, the neuronal restrictive silencer elements may be in the same transcriptional orientation W O 97/13866 PCTrUS96/16368 as the LAPI promoter, or ~It~ tively, in an opposite and divergent transcriptional orientation.
Such c~ c~ion c~settes may be utilized in a variety of constructs and methods, as described in more detail below. For example, such expression cassettes S may be delivered directly to an in vitro cell culture or to a warrn-blooded animal by a variety of techniques, including for exarnple by itself (WO 90/11092), in liposomes, condensed with polycations linked or unlinked to killed adenovirus (Curiel et al., Hurn Genc ~her. 3:147-154, 1992), or linked to a ligand (Wu etal., J. Biol. Chem 264: 16985- 16987, 198g).
GENE DELIVERY CONSTRUCTS
In addition to expression c~cettes, which are useful by themselves for transfecting cells or therapeutic purposes, the present invention also provides gene delivery constructs which are useful for carrying and/or delivering the above-described 15 ~ ession cassettes. Rc~lcscnL~live exarnples of such constructs include a variety of non-viral and viral vectors, as described below, as well as cells which are capable of producing such vectors.
For example, within one aspect of the invention retroviral vectors may be utilized as gene delivery constructs suitable for delivering the above-noted expression 20 constructs. Represelllative examples of such retroviral vectors include those described within EP 0,415,731, WO 90/07936, WO 91/0285, WO 94/03622, WO 93/25698, WO
93/25234, U.S. Patent No. 5,219,740, WO 93/11230, WO 93/10218, U.S. Patent No. 4,777,127, EP 0,345,242 and WO 91/02805).
Other exarnples of suitable gene delivery constructs may be found in the 25 Herpesvirus farnily. Briefly, suitable members of the Herpesviridae include both primate Herpesviruses, and nonprimate Herpesviruses such as avian Herpesviruses.Representative examples of suitable Herpesviruses include Herpes Simplex Virus Type 1 (McKnight etal., Nuc. Acids Res. 8:5949-5964, 1980; Fields etal., Fundamental Virolo~y, Raven Press, N.Y. ~ 1986)), Herpes Simplex Virus Type 2 (Swain and 30 Galloway, J. Virol. 46:1045-1050, 1983), Varicella Zoster Virus (Davison and Scott, J.
Gen. Yirol. 67:1759-1816, 1986) and Epstein-Barr virus (Baer et al., Nature (London) 310:207-311, 1984). Particularly preferred Herpesvirus vectors include those described below, which are deficient in the c2~lc~ion of one or more of: the virion host shut-off protein VHS; a replication loci such as IC~P~; or thymidine kinase. Representative 35 examples of such vectors include vTKhml-l, vTKhml-2 and vTKhrnl-3.
CA 02234604 l998-04-l4 W O 97/13866 PC~nUS96~I6368 Herpesviruses may be readily obtained from commercial sources such as the American Type Culture Collection ("ATCC", Rockville, Maryland). Deposits of certain of the above-identified Herpesviruses may be readily obtained fronn the ATCC, for example: ATCC No. VR-539 (Herpes simplex type l); ATCC Nos. VR-734 and S VR-540 (Herpes Simplex type 2); and ATCC No. VR-586 (Varicella Zoster Virus).
Herpesviruses may also be readily isolated and identified from naturally occurring sources (e.g., from an infected animal).
In addition to retroviral vectors and Herpes viral vectors, a wide variety of other gene delivery constructs may be utilized to deliver ~;x~lc;ssion cassettes, including for example constructs derived from adenovirus ~Rosenfeld etal., Science 252:431-434, 1991; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS
90(24):11498-502, 1993; Levrero etal., Gene 101(2):195-202, l991), and Guzman et al., Circulation 88(6):2838-48, 1993; alphaviruses such as Semliki Forest Virus and Sindbis Virus (Xiong et al., Science 243:1188, 1989; Raju and Huang, J. ~ir.
65(5):2501-2510, 1991; Hertz and Huang, J. Vir. 66(2):857-864, 1992, WO 92/10578;
WO 95/07994; U.S. Patent No. 5,091,309); influenza virus (Luytjes et al., Cell 59: 1107-1113, 1989; McMicheal et al., N. Eng J. Med. 309:13-17, 1983; and Yap et al., Nature 273:238-239, 1978); pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch etal., PNAS 86:317-321, 1989; Flexner etal., Ann. N.Y. Acad. Sci. 569:86-103, 1989; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114, 1979); parvovirus such as adeno-associated virus ~S~mnl~ki etal., J. Yir. 63:3822-3828, 1989; Plotte etal., G. Biol. Chem. 268:3781-3790, 1993; Flotte etal., PNAS 90(22):10613-10617, 1993; WO95/13365); and HI~I
(Poznansky, J. Virol. 65:532-536, 1991).
Other non viral vector systems that may also be utilized include a variety of nucleic acid based l~ s~ ion systems (e.g, based on T7 or SP6 promoters, see generally, WO 95/07994). Such vector systems may be ~-1mini~tered and prepared as described above (e.g, in liposomes, condensed with polycations, or linked to a ligand).
~EOUENCES OF INTEREST
A wide variety of heterologous nucleic acid sequences (also referred to as nucleic acid segrnent~ or molecules) may be expressed by the ~x~ ion cassettes and gene delivery constructs of the present invention, including for example, ~nti~çn~e~
CA 02234604 l998-04-l4 W O 97tl3866 PCT~US96/16368 ribozyme or regulatory sequences, as well as a wide variety of proteins such as immunogenic portions of disease-associated antigens, immunologically active molecules, replacement genes and toxic genes.
Representative examples of antisense and ribozyme sequences include 5 those sequences which inhibit, for exatnple, tumor cell growth, viral replication, or a genetic disease by preventing the cellular synthesis of critical proteins. Examples of such ~nti~nce sequences include antisense ABL (Fainstein etal., Oncogene 4:1477-1481, 1989), zinti~çn~e HER2 (Coussens et al., Science 230:1132-1139, 1985), ~nticçll~e myc (Stanton et al., Nature 310:423~25, 1984), ~nti~n~e ras and antisense CPP32 or 10 ice proteases.
Representative examples of ribozytne sequences include h~mmerhes~l ribozymes (for example, as described by Forster and Symons, Cell 48:211-220, 1987;
Haseloff and Gerlach, Nature 328:596-600, 1988, Walbot and Bruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature 334:585, 1988) and hairpin ribozymes15 (for example, as described by Haseloffetal., U.S. Patent No. 5,254,678 and Hempel et al., European Patent Publication No. 0 360 257) which have the ability to specifically target, cleave and inactivate RNA or mRNA. Briefly, the sequence requirement for the hairpin ribozyme is any RNA sequence consisting of NNNBN*GUCNNNN~ (where N*G is the cleavage site, wherein B is any of G, 20 C, or U, and where N is any of G, U, C, or A) (Sequence I.O. No. 1). The sequence requirement at the cleavage site for the h~mmerhe~-l ribozyme is any RNA sequence consisting of NUX (where N is any of G, U, C, or A and X represents C, U or A) can be targeted. Accordingly, the same target within the hairpin leader sequence, GUC, is useful for the hammerhead ribozyme. The additional nucleotides of the hammerhead25 ribozyme or hairpin ribozyme is c~et~rmined by the target fl~nkinp nucleotides and the hammerhead c~ n~en~us sequence (see Ruffner et al., Biochemistry 29:10695-10702,1990).
"Disease-associated" antigens should also be understood to include all, or various portions (e.g, immunogenic portions) of eukatyotic (including for example, 30 parasites), prokaIyotic (e.g, bacterial) or viral pathogens. Other "disease-associated"
antigens include tumor-associated antigens such as ras, p53 and CEA.
Immunologically active molecules may also be ~ essed by the expression c~ettes and gene delivery constructs described herein. As utilized within the context of the present invention, it should be understood that "immunologically 35 active molecules" refers to those molecules which can either increase or decrease the recognition, presentation or activation of a cell-mediated or humoral immune response.
CA 02234604 l998-04-l4 W O 97/13866 PCT~US96~636~
Representative examples of immunologically active molecules include Iymphokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, ~L-8, IL-9, IL-10, IL-11, IL-12 (WO
90/05147; EPO 433,827), IL-13 (WO g4/04680), IL-14, IL-15, a, 13,or ~ r~
GM-CSF, M-CSF-l, G-CSF, ICAM-1 (Simmons etal., Nature 331:624-627, 1988), 5 ICAM-2 (Singer, Science 255: 1671, 1992), 13-microglobulin (Parnes et al., PNAS
78:2253-2257, 1981), HLA Class I, HLA Class II molecules, B7 (Freeman etal., J.
Immun. 143:2714, 1989), and B7-2, as well as their respective receptors. Other biologically active molecules that may likewise be utilized in the context of the present invention include neurotrophins such as nerve growth factor (N~F), brain derived10 growth neurotrophic factor (BDGF), glial-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), tumor necrosis factor (TNF) as well as their respective receptors.
Within certain aspects of the present invention, the ~ e~sion c~etteS
and gene delivery constructs described herein may direct the expression of more than 15 one heterologous sequence. Such multiple se~uences may be controlled either by a single promoter, or ~ltern~tively~ by additional secondary promoters (e.g, Tnt~rn~l Ribosome Entry Sites or "IRES"). Within further embodiments of the invention, expression cassettes or gene delivery constructs are provided which direct the expression of heterologous sequences which act synergistically (e.g., a disease-20 associated antigen, and an immlml logically active molecule, such as IL-2, IL-12 or r-interferon).
Within other aspects of the invention, the t;~ t;ssion c~ettes or gene delivery constructs described herein may direct the expression of one or more heterologous sequences which encode "replacement" genes. As utilized within the 25 context of the present invention, the term "replacement genes" refers to a nucleic acid molecule which encodes a therapeutic protein that is capable of preventing, inhibiting, stabilizing or l~:velsillg an inherited or ncninherited genetic defect. Representative exarnples of such genetic defects include disorders in metabolism, immnne regulation, horrnonal regulation, and enzymatic or membrane associated structural function.
30 Specific examples include ~17hPimer's Disease (see, for example, Goat et al., Nature 349:704, lg91; Sherringt-n etal., Nature 375:754, 1995; Levy-Labad etal., Science 269:973, 1995) and Huntington's Disease (see EP 0 614,977 and WO 94/21790).
Replacement genes may also be ~1mini~tered for specific conditions, such as the ~(1mini~tration of anti-apoptotic genes or sequences such as Bc1-2, BclX or Bax (Oitvai 35 etal., Cell 74:60g-619, 1993), neuronal apoptosis inhibitory protein ("NAIP", Roy W O 97/13866 PCTrUS96/16368 et al., Cell 80:1-20, 1995), or TNF (Zheng et al., Nature 377:348-351, 19g5) in order to inhibit or remedy conditions or disease where apoptosis occurs.
Representative exarnples of toxic genes which may be expressed and/or delivered by the e~Le~ion csl~sett~s and gene delivery constructs provided herein include genes which encode proteins such as abrin (Wood etal., Eur. J. Biochem.
198:723-732, 1991), diphtheria toxin (Tweten et al., J: Biol. C~em. 260: 10392- 10394, 1985), antiviral protein (Barbieri et al., Biochem. J: 203:55-59, 1982; Irvin et al., Arch.
Biochem. & Biophys. 200:418-425, 1980), cholera toxin (~ek~l~nos etal., Nature 306:551-557, 1983; Sanchez and Holmgren, PNAS ~6:481-485, 1989), gelonin (Stirpeet al., J. Biol. C~em. 255:6947-6953, 1980), pokeweed (Irvin, Pharmac. Ther. 21 :371 -387, 1983), ricin (Larnb etal., E~ur. J. Biochem. 148:265-270, 1985), Shigella toxin (Calderwood et al., PN~lS 84:4364-4368, 1987), tritin, and Pseudomonas exotoxin A
(Carroll and Collier, J. Biol. Chem. 262:8707-8711, 1987).
Within other aspects of the invention, heterologous sequences should be understood to include gene products which activate a non-toxic product into a toxic product. Representative examples of such gene products include thymidine kinaseswhich activate a nucleoside analogue such as acyclovir or gancyclovir, as well as other "prodrugs" (see WO 93/10218, WO 93/01281; WO 93/08843; WO 93/08844; and WO
so/07936 ) As should be evident from the above discussion, expression cassettes of the present invention may be utilized to express a wide variety of additional sequences, including for exarnple those encoding receptors (including, for exarnple, G protein linl~ed receptors as described in more detail below), regulatory proteins, enzymes and structural proteins not specifically set forth above.
Sequences which encode the a~ove-described heterologous genes may be readily obtained from a variety of sources. For exarnple, plasmids which containsequences that encode imm~ln-~logically active molecules may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford, F.n~l~n~l). Representative sources sequences which encode the above-noted imrnune accessory molecules include ATCC No. 20663 (which contains sequences encoding alpha hlLelrel~on), ATCC Nos. 31902 and 39517 (which containssequences encoding beta hlLelr~lol1), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (which contains sequences encoding Interleukin-2), ATCC Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), ATCC No. 57592 (which contains sequences encoding Interleukin-4), ATCC Nos. 59394 and 59395 WO 97tl3866 PC~fUSg6~636~
(which contain sequences encoding Interleukin-5), and ATC(~ No. 67153 (which contains sequences encoding Interleukin-6). It will be evident to one of skill in the art that one may utilize either the entire sequence of the protein, or an a~lo~l;ate portion thereof which encodes the biologically active portion of the protein.
S Alternatively, known cDNA sequences which encode cytotoxic genes or other heterologous genes may be obtained from cells which express or contain such sequences. Briefly, within one embodiment mRNA from a cell which expresses the gene of interest is reverse transcribed with reverse trans.~ se using oligo dT or random primers. The single stranded cDNA may then be amplified by PCR (see U.S.
Patent Nos. 4,683,2~2, 4,683,195 and 4,800,159. See also PCR Technology: Principles and Applications ~or DNA Amplification, Erlich (ed.), Stockton Press, 1989 all of which are incorporated by reference herein in their entirety) lltili~ing oligonucleotide primers complementary to sequences on either side of desired sequences. In particular, a double stranded DNA is denatured by heating in the presence of heat stable Taqpolymerase, sequence specific DNA primers, ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA.
Sequences which encode the above-described genes may also be synth~i7~-1, for example, on an Applied Biosystems Inc. DNA synth~?si7~r (e.g., ABI
2~ DNA synth~ r model 392 (Foster City, California)).
G PRoTElN-LINKED RECEPTORS AND RECOMBINANT HE~PESVIRUS VECTORS
Within the various aspects of the present invention, the expression c~settes or gene delivery vehicles described herein (e.g, recombinant Herpesvirus vectors may be utilized as a means of introducing nucleic acid segments into nonrnitotic cells primarily of the nervous system (collectively referred to as "neural" or "neuronal"
cells). For example, recombinant He,l,e~vi,us vectors of the present invention may be utilized to deliver nucleic acid segm~nt~ into the cell where the proteins are expressed, generally as mRNA which is then tr~n~l~te~l into a protein. When the protein tr~n~l~tecl 3~) is a G protein linked receptor, for example, the 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 me~nger system and, thus, function in much the same manner as a naturally occurring receptor.
- Briefly, Herpesviruses such as HSV-I are double stranded DNA viruses 35 (approx. 152 kb) which are replicated and transcribed in the nucleus of the cell.
Although HSV-l is utilized as a representative Herpesvirus in the description and examples provided below, it should be understood that the present invention is not so limited. In particular, numerous other Herpesviruses (including for exarnple~ those described above), may be utilized within the context of the present invention.
Productive infection by HSV-l usually results in cell Iysis or alteration 5 of host macromolecular processes. However, HSV~1 also may be m~int~ined in~lefinitely in the "latent state" in certain cells by a mechanism involving the tegument of the virus particles. The reactivation of the virus is regulated by certain systemic or cellular events. The latent virus is still transcriptionally active, producing "latency associated transcripts" (LATS). Mutant viruses that are compromised or defective in 10 their replication potential can still enter the latent state (e.g., UL41(-), TK(-), and ICP4(-)). In fact, a TK(-) HSV-I will mslint~in the latent state in~efnitely. Thus, HSV-I is ideal for use in delivering nucleic acid segments to non-mitotic cells such as neuronal cells. Within the present invention, Herpesvirus vectors such as those derived from HSV-I are preferably m~mt~inP!d in the latent state.
The manipulation of Herpesviruses such as ~ISV-I for the purposes of the present invention, may, within certain aspects involve deletions, substitutions or mutations of non.?~c~nti~l regions of a Herpesvirus genome, generally m;.;~L~ir~i"~ the essential regions intact. In the context of the present invention, "essenti~l region" refers to any region of the viral genome the deletion of which would result in an inability to 20 infect a m~mm~ n host cell or an inability to replicate, even with the assistance of a helper virus or a complementing cell line. Non~?sct?nti~l 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 re~uired for 25 the propagation of replication defective viruses that can not, by defînition, propagate themselves. Such helper viruses are described in Fields et al., Fundamental Virology, Raven Press, N.Y. (1986), and are well known to those skilled in the art. Examples of helper viruses suitable for use in the present invention include unaltered Herpesviruses such as HSV-l as well as other viruses that express the genes contained within the 30 deleted region whose products are nec~ ry for propagation of a recombinant Herpesvirus.
The terrn "complementin~ cell lines" refers to cell lines that provide gene products re~uired for the propagation of defective viruses that by definition cannot propagate themselves. Suitable complem~ntin~ cell lines in the present invention35 include E5 Vero cells, which provide the protein ICP4 for replication deficient viruses.
(Disclosed in detail in DeLuca et al., J. Yir. 56:558-570, 1985.) CA 02234604 l998-04-l4 W O 97~13866 PCT~US96/I6368 As noted above, within certain aspects of the present invention, nucleic acid segments are inserted into a Herpesvirus genome and/or portions of the Herpesvirus genome are deleted. Preferably, insertions or deletions of nucleic acid segments utilized in the present invention are made to one or more of the following 5 nonessential regions: the UL41, thymidine kinase (TK), and/or any one of several replication loci (Ward and Roizman, Trends in Gen 10:267, 1994). The replication loci include DNA polymerase and that for the ICP4 protein. Briefly, ~CP4 is a proteinproduced by a viral immediate-early gene and governs transcriptional regulators required for the e2.~lc~ion of the early genes. Likewise, Thymidine kinase is an early 10 gene implicated in the replication of viral DNA. UL41 is a late gene whose protein product is responsible for early shut off of host cell macromolecular synthesis.The Herpesvirus genome can be manipulated to produce such deletions and insertions by using standard recombinant DNA techniques, such as those described in Maniatis etal., Molecular Cloning, ~ Laboratory Manual, Cold Spring Harbor 15 Laboratory, Cold Spring Harbor, N.~. (1982), or Sambrook et al., Molecular Cloning, A Laboratory Manu~l, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). Briefly, deletions within a Herpesvirus genome can be effected by conventional techniques employing endonucleases, exonucleases and the like. Insertions can also be executed using conventional techniques, including, by way 20 of example cotransfection, i.e., homologous recombination facilitated by a suitable plasmid. A suitable plasmid available for such use includes pl~c/CMV (Invikogen Corp.). The plasmid incll-r1ing the desired characteristics can be selected using conventional methods and introduced for propagation purposes into a host cell ororganism using standard transformation procedures. The plasmid is then isolated from 25 the host org~ni~m, mixed with unaltered Herpesvirus DNA and cotrzln~fecte~ into host cells. The cells cont~inin~ the plasmid and the Herpesvirus DNA are cultured, and homologous recombination take place, resulting in the replacement of the unaltered region in the viral DNA with the corresponding altered region from the plasmid. Any host cell suitable for plasmid and ~erpesvirus DNA transfection and subsequent 30 recombinant virus propagation can be utilized in this procedure. The recombinant Herpesvirus DNA is then replicated within the cell and the viruses which have undergone the desired recombination are selected using standard techniques.
As noted above, recombinant Herpesvirus of the present invention are produced through insertion of nucleic acid segments into the genome. Within the 35 context of the present invention, "nucleic acid segment" refers to a nucleic acid sequence or molecule, and may be derived from a variety of sources including DNA, CA 02234604 l998-04-l4 W O 97/13866 PCT~US96/16368 cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid segments may comprise genomic DNA which may or may not include naturally occurring introns.
Such genomic DNA may be obtained in association with promoter regions or poly A
se~uences. Further, The nucleic acid segment may be aIl antisense sequence. The 5 nucleic acid segments of t~e 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 any one of several means. Alternatively, mRNA can be isolated from a cell and used to produce cDNA ~oy reverse transcriptase by any one of several methods.
Within particular prefe~red embo-~iment~ of the present invention, the nucleic acid se~ment is a G protein linked receptor gene. In the context of the present invention, the term "G protein linked receptor" refers to a gua~ine nucleotide binding regulatory protein coupled to both a cell surface receptor and an effector, such as an ion channel, together comprising a tr~n~memhrane .~i~n~lin~ system. G protein linked15 receptors me~ t~ the actions of extracellular signals, such as neurotr~n.~mitt~rs. They are described in detail in Dohlman etal., Ann. Rev. Bioc*em. 60:553-588 (1991~.
Suitable G protein linked receptors genes include those listed in Table I and portions thereof.
It will be evident to those skilled in the art that the particular receptor 20 utilized will be influenced by the characterist;cs of the receptor and the specific tre~tm~nt Receptor Subtype Species Ref.
M~m~
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Rat Machida, C.A., et al., J. Biol. Chem. 265:12960-65, 1990.
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Human Kobilka, B.K., et al., Proc. Natl. Acad Sci. USA
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Schofield, P.R., et al., Nucleic Acid~ Res. 15:3636, 1987.
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Emorine, L.J., et al., Proc. NatL Acad Sci. USA
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Mouse Allen, J.M., et al., EMBO J. 7:133-38, 1988.
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Ml-muscarinic Pig Kubo,T.,etal.,Nature323:411-16, 1986.
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M2~ u ,cali~ic Pig Kubo, T., et al., FEBSLett. 209:367-72, 1986.
Peralta, E.G., et al., Science 236:600-5, 1987.
Human Peralta, E.G., et al., EMBO J. 6:3923-29, 1987.
Rat Gocayne, J., et al., Proc. Nat'l. Acad. Sci USA
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Bonner, T.I., et al., Science 237:527-32, 1987.
M3-~llu~ alillic Human Peralta, E.G., et al., EMBO J. 6:3923-29, 1987.
Rat Bonner, T.l., et at., Science 237:527-32, 1987.
M4-muscarinic Human Peralta, E.G., et al., EMBO J. 6:3923-29, 1987.
Rat Braun, T., et al., Biochem. Biophys. Res. Commun.
149:125-32, 1987.
Pig Akiba, l., et al., FEBS Lett. 235:257-61, 1988.
M5-muscarinic Human Bonner, T.l., Neuron. 1:403-10, 1988.
Rat Bonner, T.I., Neuron. 1:403-10, 1988.
Liao, C.F., et al., J. Biol. Chem. 264:7328-37, 1989.
Dl-dop~minPrgic Human Dearry, A., et al., Nature 347:72-75, 1990.
Zhou, Q.Y., et al., IVature 347:76-80, 1990.
Rat Zhou, Q.Y., et al., Nature 347:76-80, 1990.
O'Dowd, B.F., et al., FEBSLett. 347:8-12, 1990.
D~-dopaminergic Rat O'Dowd, B.F., et al., FEBSLett. 347:8-12, 1990.
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Human Todd, R.D., et al., Proc. Nat'l. Acad. Sci. USA
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D3-dopaminergic Rat Sokoloff, P., et al., Nature 347: 146-51, 1990.
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Human Gerard, N.P., et al., J. Biol. Chem. 265:20455-62, 1 990.
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Thyrotropin Dog Pral,~.e.. Lic~, M., etal., Science246:162û-22, 1989.
Libert, ~., et al., Mol. Cell. Endocrinol. 68:R15-17, 1990.
Human Libert, F., et al., Biochem. Biop*ys. Res.
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CA 02234604 l998-04-l4 WO 97/138G6 PCT~US96fI6368 Green opsin Human Nathans, J., et al., Science 232: 193-202, 1986.
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Although it is preferable to utilize the complete coding sequence from the G protein liinked receptor gene, within certain embodiments of the invention only that portion of the G protein linked receptor gene which encodes expression of the 5 receptor on the cell surface need be utili7~o~1 Within the context of the present invention, both the entire coding region and portions thereof are referred to as "G
protein linked receptor genes." Such ex~ ion can be ~letermin~l by any one of several suitable means, including ligand binding assays.
The coding sequence for the G protein linked receptor should be inserted in such a manner that the resulting recombinant Herpesvirus genome directs the synthesis of a L~ sc.ipt that is capable of being trRn~ t( ~1 into a G protein linked receptor protein. The desired G protein linked receptor produced should be compatible S with Herpesvirus propagation (i.e., is not lethal). The promoter sequence can be supplied within a separate or the same nucleic acid segm~nt as the G protein linked receptor sequence or by the genomic portion of the recombinant virus. Suitable promoters include any one of several which are capable of initiating expression of the G
protein receptor gene. Preferably, the promoter is a major immediate early promoter 10 and the sequence includes a polyadenylation site. More preferably, the promoter is the cy~omegalovirus (CMV) promoter.
In a preferred embodiment of the present invention, the Herpesvirus utilized is deficient for ~ t;ssion of the thymidine kinase (TK) gene locus (TK(-)).
More preferably, the G protein linked receptor sequence is inserted in the thymidine 15 kinase (TK) gene locus of an HSV-l genome, rendering it deficient. Within the context of the present invention, "deficient" refers to low or nonexistent expression of the gene in question. Deficient expression generally results from insertion into or deletion of the genet~c loci in question. Deficiency of the thymidine kinase loci can be assayed using any one of several means, including selection with bromodeoxyctidine using standard 20 methods (see also Surnmers et al., PNAS 72:4081, 1975).
In another preferred embodiment of the present invention, the Herpesvirus genome ;s deficient for the expression of virion host shut off gene (IJL4 1 ) locus and the thymidine kinase (TK) gene locus. Even more preferably, a nucleic acid segment encoding beta-galacto~ e is inserted in the virion host shut-off gene (UL41) 25 locus to allow for easy confirmzltion of successful debilitation and the G protein linked receptor sequence is inserted in the thymidine kinase (TK) gene locus. The deficiency in UL41 ~ression may be assayed for by ~letecting beta-galactositl~e expression using standard techniques (see also Smibert et al., J. Vir. 68:2339, 1994).
In another aspect of the present invention, the Herpesvirus genome is 30 a~ditionally deficient in the expression of a viral gene required for replication ("replication deficient"). Briefly, proteins required for replication include, by way of example, ICP4 and DNA polymerase. Preferably, it is replication deficient in thes~ion of the ICP4 protein. Replication deficiency can be assayed using any one of several standard methods, including by comparison of cultures in complementary and 35 noncomplementary cell lines (see also DeLuca et al., J. Vir. 56:558-570, 1985).
wc~ s7rl3s66 PCT~US96~16368 In another embodiment of the present invention, HSV-l 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 UL4 1 loci and ICP4 protein.
Within the context of the present invention, "vTKhml-l" refers to a recombinant Herpesvirus vector which is deficient in both the expression of the viral host shut offprotein (VHS) and thymidine kinase (TK). (FIG. lb); "vTKhml-2" refers to a recombinant Herpesvirus vector which is deficient in the expression of both the viral transcriptional regulator (ICP4) and thymidine kinase (TK). (FIG. lc); and"vTKhml-3" refers to a recombinant Herpesvirus vector which is deficient in both the e~x~le~SiOn of the viral transcriptional regulator, ICP4, VHS, and thymidine kinase (TK). (FIG. ld). All three of the recombinant viruses express a G protein linkedreceptor (preferably inserted in the TK locus) from an imm~ te early promoter, preferably a CMV promoter. As described in more detail below, these recombinant Herpesvirus vectors are characterized by low cytopathicity and a high rate of expression. Recombinant Herpesvirus vectors with "çssenti~lly the same ch~r~cteristics"
is intended to refer to recombinant Herpesvirus vectors with the same or similardeficiencies in expression.
These and other recombinant Herpesvirus vectors characterized by low cytopathicity and/or a high level of ~ ion of G protein linked receptor may be produced by cllltllring a first and second recombinant Herpesvirus in a suita~le cell line for a time sufficient and under suitable conditions to allow for recombination. The first recombinant Herpesvirus is one carrying a G protein linked receptor gene and capable of expression thereof and the second recombinant Herpesvirus is replication deficient.
The G protein linked receptor nucleic acid segment may be inserted into the first recombinant Herpesvirus by any suitable means described above, including homologous recombination between the virus and a plasmid carrying the G protein linked receptor nucleic acid segment Recombinant Herpesvirus vectors carrying the G
protein linked receptor sequence may then be selected for using standard methods, including restriction digestion followed by Southern Blot hybridization. Preferably, the first recombinant Herpesvirus is TK(-) HSV-l. Even more preferably, the G protein linked receptor gene is inserted in the TK locus of the first recombinant Herpesvirus.
Additionally, the first recombinant Herpesvirus is preferably deficient in expression in the virion host shut-off protein (VHS). Most preferably, the first recombinant virus is vhsA (J. Smiley, McMaster U~livelsiLy, Hamilton Ontario) (FIG. lA). Briefly, vhsA is a mutant HSV-l which bears the beta-galactosidase gene in the UL41 region of its CA 02234604 l998-04-l4 W O 97/13866 PCT~US96/16368 genome, rendering it deficient in expression of the virion host shut-off protein. The G
protein linked receptor gene may be inserted into vhsA by the means described above.
Preferably, the second recombinant Herpesvirus vector is replication deficient. Even more preferably the second recombinant Herpesvirus vector is def1cient in the expression of the ICP4 protein. Most preferably, the second recombinant HSV-1 is dl20. (Disclosed in detail in DeLuca etal., "Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Irnmediate ~arly Regulatory Protein ICP4," ~ Vir. 56:558-570, 1985). Briefly, dl2Q is replicationdeficient HSV-l, due to 11iminisht-d ~ ession of ICP4. Recombinants defective for 10 ICP4 expression may be selected using any one of several suitable methods noted above including Southern blot analysis, Northern blot analysis, or immunofluorescence studies.
If both the first and the second recombinant Herpesviruses are replication deficient, the two recombinant Herpesviruses can be transfected on a complementary 15 cell line for replication. Suitable complement~ry cell lines include E5 Vero cells (ICP4(+)). (Disclosed in detail in DeLuca etal., "Isolation and Chara~ ion ofDeletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Immediate EarlyRegulatory Protein ICP4," J. Vir. 56:558-570, 1985).
The recombinant Herpesvirus vector resulting from the transfection of 20 the first and second recombinant Herpesviruses may be selected for one or more of four basic characteristics: (1) thymidine kinase deficiency, (2) ICP4 expression, (3) UL41 ~xp~e~ion, and (4) G protein receptor gene ~p.~,s,ion, using any one of several suitable methods described above. By way of example, thymidine kinase expressioncan be screened for using bromodeoxycytidine; ICP4 expression can be screened for :25 based on the virus' ability or inability to grow on the complementin~ cell lines; UL41 expression can be screened for based on beta-g~ rt~idase production, and t;~lt;ssion of the G protein linked receptor gene can be screened for based a on ribonuclease protection assay. Maniatis etal., Molecular Cloning, ~ Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Thus, three ple~-~t;d30 embodiments of the invention vTKhml-l (FIG. lb~, vTKhml-2 (FIG. lc), and vTKhml-3 (FIG. ld) may be produced and screened a~cording to ~2~ple~ion. The more preferred embodiment is vTKhml-3 (FIG. ld).
As noted above, within other aspects of the present invention, recombinant Herpesvirus vectors can be used to deliver G protein linked receptor~5 nucleic acid sequence to mslrnm~ n cells. Once infected, the recombinant Herpesvirus vector will then produce the desired receptors which are expressed on the cell surface.
, WO g7/13866 PCT~US9Cf~6368 The infected cells are then selected for the desired G protein linked receptor expression.
For virus infection, the recombinant Herpesvirus vectors may be applied to the cells under standard cell culture conditions. Cell culture techniques are described in Maniatis et al., Molecular Cloning, ~4 Laboratory Manual, Cold Spring Harbor Laboratory, Cold 5 Spring Harbor, N.Y. (1982~. The specific host cells employed in the present invention are not critical as long as they allow replication and ~pl~s~ion of the recombinant Herpesvirus vectors. Suitable cells include Vero cells (ATCC Accession No. CRL
1587).
To select for the t;~ t;s~ion of G protein linked receptors, standard 10 techniques may be employed, including ribonuclease protection assays such as those described in Maniatis et al., Molecular Cloning, ,4 Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Briefly, a labeled RNA probe is synthesi7~-1 that is partially complemcnt~ry to the region of the target mRNA. This labeled RNA probe is added to samples of the total cellular RNAs isolated from the cell 15 culture after post infection by the recombinant virus. The mixture is incub~te~l, for a sufficient time and under suitable conditions to enable a labeled probe to hybridize to the complementary RNAs and then subjected to digestion by suitable restriction enzymes, such as RNase A and RNase Tl. Labeled probe that hybridized to complementary transcripts will be protected from digestion and may be separated on a 20 polyacrylamide gel and viewed by autoradiography.
G linked protein receptor e~ ion resulting from the insertion of the recombinant viruses of the instant invention into the cell can be detected using any one of several methods known in the art, including for example, ligand binding assays.
Representative ligand binding assays suitable for use within the present invention 25 include those described in Conn, Methods in Neurosciences (Vol. 9), "Gene Expression in Neural Tissues" Academic Press, Inc., San Diego, ~alifornia (1992). For example, within one embodiment the cells infected with the recombinant virus are incubated with a radiolabeled antagonist. Saturation curves may then be performed in order to determine the approximate number of receptors ~represented by counts measured using 30 the antagonist and competitive inhibition). Within other embo-limentc, stimulation of second messenger systems maybe be ascertained by any one of several suitable means, including, for example, phosphatidylinositol (PI) turnover assays.
The recombinant viruses of the present invention may be characterized in a variety of manners, including for example, by the number of receptors expressed on 35 cells infected with the virus, the in vivo cytopathicity of the virus, and/or the immlln~genici~y of the virus. For example, within certain embodiments of the present CA 02234604 l998-04-l4 W O97/13866 PCT~US96/16368 invention, recombinant Herpesvirus vectors are provided which express greater than about 10,000 receptors per cell, preferably between about 25,000-200,000 receptors per cell, more pre~erably greater than about 200,000 receptors per cell and n1ost preferably between about 200,000 to 400,000 receptors per cell, or even greater than about 400,000 5 receptors per cell. Within other embodiments, recombinant viruses are provided which have an in vivo cytopathicity of general}y less than the in vitro cytopathicity."Cytopathicity" as used herein, refers to cell survival five days after infection.
Cytopathicity may be measured using any one of a wide variety of techniques known in the art, including commercially available kits. Suitable kits include Live/DeadTM
10 (Molecular Probes Inc.; viability/cytotoxicity kit lltili7ill~ a method of staining).
By way of example, vTKhml-1 is characterized by a surface receptor expression rate generally greater than 10,000 receptors per cell; typically in about the range of 60,000 to 80,000; and preferably in about the range of 70,000 to 100,000.
vTKhml-2 is characterized by a cytopathicity of generally less than 15 50%; typically in about the range of 35%-40%; and preferably in about the range of 20%-35%. vTKhm1-2 is further characterized by surface receptor expression generally greater than 80,000; typically in about the range of 120,000 - 160,000; and preferably in about the range of 160,000 to 200,000.
vTKhm1-3 is characterized by a cytopathicity of about in vi~ro ~0 cytopathicity of less than about 3%, typically in about the range of 0.1% to 1.0% aIld preferably in about the range of .001% - 0.1%. vTKhm1-3 is further characterized by surface receptor expression generally greater than 800,000 receptors, typically in the range of 1-1.5 million receptors, and preferably 1.25-2 million receptors/cell.
ADDITIONAL METHODS AND COMPOSITIONS
Within other aspects of the invention, methods are provided for producing a protein of interest, comprising the steps of introducing an t;~ e~ion cassette or gene delivery construct as described above, into a host cell, and culturing the hot cell under conditions, and for a time sufficient to permit ~xplc~ion of the protein.
3~) The expression cassettes may be introduced by a wide variety of mech~ni~m~, including for example, including for example calcium phosphate-mediated transfection (Wigler et al., C~ 4:725, 1978), lipofection; gene gun (Corsaro and Pearson, Somatic Cell Gen.
7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (NeumRnn et al., EMBO J. I:841-845, 1982), protoplast fusion-mediated transfection or DE~QE-dextran me~liRt~d transfection (Ausubel et al., (eds.), Current Protocols in Molecu~ar Biology, John Wiley and Sons, Inc., NY, NY, 1987).
=
WO 97~3866 PCT~US96~16368 Optionally, the resultant protein may be purified by a variety of methods, including for example, within one embodiment cell super~t~nt may be first concentrated using cominercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following concentration, the 5 concentrate may be applied to a suitable purification matrix, or ~Itern~tively, purified utilizied anion or cation çxeh~n~e resins. Finally, one or more reversed-phase high olll.ance liquid chromatography (RP-HPLC) steps may be employed.
Expression cassettes or gene delivery constructs of the present invention may also be utilized to introduce a selected sequence of interest into an in vitro culture 10 cont~ininP neuronal cells, comprising the step of introducing an expression cassette or gene delivery construct, as described herein, into an in vitro culture of cells cont~ining neuronal cells.
In another aspect of the present invention, and using the techniques described above, the gene delivery constructs or recombinant viruses of the present 15 invention can also be packaged in a suitable cell line. For example, within one embodiment of the invention, recombinant HSV-I is cultured ex vivo in suitable m~mmz~ n cells. These cells may then be introduced in vivo, using the techniquesdescribe below, i.e., stereotactical microinjection, for l~e~l...cnt of neurological disorders or analysis. Alternatively, the t:x,~les~ion cassette or gene delivery construct 20 may be introduced directly in vivo by any one of several methods described below.
In another aspect of the present invention, the ~x~-~s~ion cassettes or gene delivery constructs described above are a-lminict~red to a warm-blooded animal (e.g, a human, monkey, cow, sheep, dog, cat, rat or mouse), or other-type of animal (e.g, fish) for the tre~trnent of neuronal cell disorders, in both the central and peripheral 25 nervous system. Such c~ett~s or constructs may be utilized in the tre~tment of a wide variety of disorders, including for example, brain turnors, degenerative disorders, neural disorders characterized by abnormal gene expression, and inherited disorders.
The ~ s~ion cassettes or gene delivery constructs of the present invention may also be utilized to deliver normal genes. This allows for the tre~tment of 30 deficiency state disorders, usually of enzymes, by increasing production thereof.
Additionally, the recombinant 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 ~ s~iv~ disorders. For example, expression c~ettes of the present invention may be utilized to express sequences of interest in non-human 35 transgenic ~nim~ such as mice, rats, rabbits, sheep, dogs and pigs (see Hammer et al.
(Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), Brinster et al.
W O 97/13866 PCTrUS96tl6368 (Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985), Palmiter and 13rinster (Cell 41:343-345, 1985) and U.S. Patent No. 4,736,866). For example, within one embodiment anexpression c~sette may be introduced into pronuclei of fertilized eggs, for example, by microinjection. Integration of the injected DNA may be detected by blot analysis of S DNA from tissue samples. It is pl~rcl.~d that the introduced DNA be incorporated into the germ line of the animal so that it is passed on to the animal's progeny. Such techniques allow for, within preferred embodiments, tissue-specific (eg., neuronai cell) ession of a desired sequence of interest.
The ~x~-~s~ion c~ette~ or gene delivery constructs of the present 10 invention may also be used to create nnh~l~nced state disorders involving structural or regulatory proteins, in a model system, which could be used in efforts to establish and study methods of counteracting the effect of the imh~ nce.
In one aspect of the present invention, ~x~iei,~ion r~settes or gene delivery constructs may be used to treat neurodegenerative disorders including, by way 15 of exarnples, Alzheimer's ~ ez~ce~ Spinal muscular Ataxia, mytonic dystrophy, Spinal Balbar Muscular Atrophy (SBMA), Kennedy syndrome, Parkinson's disease, Senile dementia, Circllln~ribed cerebral atrophy, Hlmtin~tQn's chorea, Cerebrocerebellar degeneration, Amaurotic family idiocy, Leukodystrophy, Familial myoclonus epilepsy, Hallervorden-Spatz ~li.eç~ç, Wilson's ~ice~ce, hepatolenticular degeneration, Westphal-20 Strumpell pseudosclerosis, Paralysis ~itsln~, Dystonia musculorum deformans, torsiondystonia, Hallervorden-Spatz ~i~ç~ce, Spasmodic torticollis, Cerebellar degenerations, Spinocerebellar degenerations, Friedrich's ataxia, Marie's hereditary ataxia, Amyotrophic lateral sclerosis, Progressive muscular atrophy, Progressive bulbar palsy, Primary lateral sclerosis, Werdnig-Hoffmarm disease, Wohlfart-Kugelberg-Welander25 syndrome, Hereditary spastic paraplegia, Progressive neural m~lcc~ r atrophy, Peroneal muscular atrophy (Charcot-Marie-Tooth) Hypertrophic hll~ ial neuropathy (Dejerine-Sottas), Leber's disease, retinitis pigmentosa and fragile X disorder.In another aspect of the present invention, t;x~fcs~ion cassettes or gene delivery constructs may be used to treat disorders char~cteri7e~1 by abnormal gene 30 expression, and inherited disorders caused by a known gene defect. In addition to a number of the disorders listed above, genes for defective enzymes or proteins have been identified, by way of example, for (1) Iysosomal storage disorders such as those involving ,B-hexosarninidase (Kornerluk etal., J. Biol. Chem. 261:8407-8413, 1986);
Myerowitz etal., Proc, Natl. Acad. Sci. flJSA) 82:5442-5445, 1985); Tsuji etal., N.
35 ~ngl. ~ Me~. 316:570-575, 1987), (2) for deficiencies in hypoxzlntlline phosphoribosyl transferase activity (the "Lesch-Nyhan" syndrome; Stout et al., Met. }inzymol. 151:519-=
WC~ 97J~3866 PCT/IJS96J16368 530, 1987), (3) for amyloid polyneuropathies (prealbumin; Sasaki et al., Biochem.
Biophys. Res. Commun. 125:636-642, 1984), (4) for ~17heimer's Disease (see, for example, Gont etal., Nature 349:704, 1991; Sherrington etal., Nature 375:754, 1995;
Levy-Labad etal., Science 269:973, 1995; Tanzi etal., Science 235:880-884, 1987;5 Goldgaber et al., Science 235:877-880, 1986), (5) for D-lrhenne~s muscular dystrophy (uncharacterized muscle protein; Monaco et al., Nature 323:646-650, 1987), and (6) for retinoblastoma (uncharacterized protein expressed in the retina and other tissues, Lee et al., Science 235:1394-1399, 1987; Friend et al., Nature 323:643-646, 1986).
Expression cassettes and gene delivery constructs may also be used to 10 study the "shiverer" mutation (myelin basic protein, Roach etal., Cell 42:149-155 (1987); Molineaux etal., Proc. Na~l. Acad. Sci. (USA) 83:7542-7546 (1986), and the "jumpy" mutation (proteolipoprotein, Nave et al., Proc. Natl. Acad. Sci. fUSA) 83:9264-9268 (1986); Hudson et al., Proc. Natl. Acad. Sci. fUS~l) 84:1454-1458 (1987)).
Expression cassettes and gene delivery constructs of the present 15 invention can also be used for trç~tment of acute injuries to the brain or peripheral nervous tissue, for exarnple from a stroke, brain injury, or spinal cord injury.Expression ç~c~ett~s and gene delivery constructs of the present invention may also be used in the ~r~ .cnt of disorders which require receptor modulation to increase or decrease tr~nsmit~çr uptake. Such disorders include 20 schizophrenia, obsessive-compulsive disorder, depression, and bipolar mood disorders.
As utilized within the context of the present invention, the term "trç~tment" refers to re~lucing or alleviating ~y~ OlllS in a subject, preventing symptoms from worsening or progressing, inhibition or elimin~tion of the causative agent, or prevention of the infection or disorder in a subject who is free th..~rl.~lll.
25 Thus, for example, tre~tment of infection includes destruction of the infecting agent, inhibition of or hllelr~l~ .lce with its growth or maturation, neutralization of its pathological 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 unb2l~nce~1 state disorder is "treated" by partially or wholly remedying the 30 imb~l~nce which causes the disorder or which makes it more severe.
The ~x~r~ssion c~ettes or gene delivery constructs of the present invention may be ~1mini~t~red by any one of several methods of ~-lmini~tration known in the art which account for the risk of degradation of the recombinant virus in the bloodstrearn and such that the virus retains its structure and is capable of infecting target 35 cells. Within one embodiment, ~lmini~tration may be accompli~hed by microinjection of the virus, alone or in a pharmzl~eutir~lly suitable carrier or diluent, through a CA 02234604 l998-04-l4 W O97tl3866 PCT~US96/16368 stereotactically-located pipette or syringe. Suitable locations vary with application, but include intraocular and brain injections.
Phar~m~ceutical carriers and diluents which are suitable for use within the present invention include, for example? water, lactose, starch, m~gne~iurn stearate, talc, 5 gum arabic, gelatine, polyalkylene glycols (e.g., polyethylene glycol), and the like. The ph~rmslreutical preparation 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 ph~rm~reutical p.c~aldLions can be subjected to 10 conventional ph~rm:~celltical adjuvants such as preserving agents, stabilizing agents, wetting agents, salts for varying the osmotic ~les~ t;, and the like. The present pharmaceutical ~ ualdlions may also contain other therapeutically valuable substances.
In another aspect of the present invention, ~r~ssion cassettes or the delivery constructs described herein may be delivered by chronic infusion using any 15 suitable method known in the art, including an osmotic minipump (Alza Corp.) or delivery through a time release or sll~tzlinecl release medium. Suitable time release or sl-ct~ine~l release systems include any methods 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 20 meçh~ni~m~ the composition may be injected into the cerebrospinal fluid via intrathecal or intraventricular injections, as well as into the brain substances and intraocular locations.
The expression cassette or gene delivery construct should be 7~lmini.ctcred in a therapeutically effective amount. A therapeutically effective amount 25 is that sufficient to treat the disorder. A therapeutically effective amount can be determined by in vitro ~ in~ent followed by in vivo studies. Expression of the inserted nucleic acid segment can be deterrnined in vitro using any one of the techniques described above. Expression of the inserted nucleic acid segment can be ~etermined in vivo using any one of several methods known in the art, including 30 immllnofluorescence using a fluoresceinated ligand.
In another aspect of the present invention, the t;~les~ion cassettes or gene delivery constructs described above may be incorporated into a phslrrnzlceutical composition. Preferably, the ph~ reutical composition contains one or more therapeutically effective doses of the cassette or construct in a suitable ph~rm:~relltical 35 carrier or diluent. Suitable pharmaceutical carriers and diluents are outlined above. A
theldpeuLically effective dose may be determined by in vitro ~p~ ent followed by W O 97/I3866 PCT~US96~16368 in vivo studies as described above. The composition may be ~tlmini~tered by any one Or the methods described above.
The following examples are provided by way of illustration, and not by way of limitation. Unless otherwise incli~t~-~, the specific protocols used in the 5 following examples are described in detail in Maniatis et al., supra, or Sambrook et al., supra.
E~AMPLE 1 GENERATION OF A RECOMBlNANT HERPESVIRUS VECTOR
1 0 WHIC~I EXPRESSES M 1 MUSCARINIC ACETYLCHOLINE RECEPTOR
A recombinant Herpesvirus vector which expresses the ml m-lc~rinic acetylcholine receptor (ml-AchR) was generated by homologous recombination between an HSV-l virus and a plasmid, pTKhml, which was constructed for this 15 purpose.
Briefly, pTKhml was prepared from the coding sequence for the hurnan ml-AchR gene and altered pTKSB. The coding sequence of ml-AchR was isolated as a 2.7 kb BamHI fragment from a plasmid cont~ining this sequence (Bonner et al., Science 237:527-532, 1987; see also generally Genbank Accession Nos.: M16404, 20 M16405, M16406, M16407, M16408, M16409) and inserted into a plasmid vector cont~ining a single BamHI cloning site. The coding sequence was re-isolated by digestion of that plasmid vector with EcoRI and HindIII.
pTKSB (Smiley et al., J. Vir. 61(8):2368-77 (1987)), which contains the HSV-I TK gene, was altered by insertion of a CMV promoter-co.l(~;..i..g fragment25 from the plasrnid pRc/CMV (Invitrogen Corporation). This fragment r~lt;s~llts the portion of the plasmid ~xtenfling from base 2û9 to base 1285 and co~ g the CMV
major imm~ te early promoter, a multicloning site, and a poly A addition site. The fragment was inserted into pTKSB by first digesting the plasmid with BamHI and then converting the BamHI site into a PacI site by the addition of adapter sequences. The 30 CMV promoter was oriented in the opposite direction to the TK promoter to reduce llallsc~ ional interference. The resulting plasmid (pTKSB cont~ining the CMV
promoter) was then digested with EcoRl and HindIII and ligated to the ml-AchR
coding se~uence which had also been digested with HindIII and EcoRl using collv~l,tion~l methods. This plasmid was referred to as pTKhml.
3~ pTKhml was then used to generate an HSV recombinant virus by in vivo homologous recombination. pTKhm l was cotransfected into Vero cells (ATCC
W O 97/13866 PCT~US96116368 Accession No. CRL 1587) along with an infectious HSV-1, vhsA. vhsA is a mutant HSV-l (FIG. la) ~see Smibert and Smiley, J: Vir. 64:3882, 1990) con1~inin~ the ,B-galactosidase gene in the UL4 1 gene coding sequence.
TK deficient recombinants were selected using bromodeoxycyt;dine.
S Following selection, virus isolates were plaque purified and tested for the CMV-ml-AchR insert by digestion with EcoRI, electrophoresis on a 1.1% agarose/TAE gel and hybridization to a radioactive probe. The probe was generated by incubating the mlAchr gene in buffer contzlining random hexamers of DNA to act as primers for extension 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 hybridization at 37~C in the presence of 50% formamide, 2X standard saline citrate, 5X Denhardt's solution, 1%
sodium dodecyl sulfate. Following incubation for 12 h, filters were washed extensively 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. lb), lacked a 2.1 kb EcoRI fragment Contzlinin~;
the endogenous TK gene and instead, contained a 4.6 kb EcoRI fr~mPnt which hybridized to the m 1 -AchR specific probe. Thus, it was det~rmin~cl that the neurotransmitter receptor gene was successfully introduced into the viral genome.
EXAMPI,E 2 DETECTION OF M 1 AcHR MRNA EXPRESSION FROM
RECOMBINANT VIRUSES
Expression of ml AchR transcripts from the CMV promoter was detected using a ribonuclease protection assay. (FIG. 2). Briefly, a labeled RNA probe was synthesized from 326 nucleotides (nt) from the T7 promoter of the plasmid BS/KS(-) (available from Stratagene Cloning Systems) comprising 265 nt of the 5' end of the mlAchr gene and ~6 nt of the 3' end of the CMV promoter. This probe targeted the 5' end of hurnan mlAchR mRNA as well as a portion of the CMV promoter. This labeledprobe was incubated with sarnples of total cellular RNAs isolated from Vero cells 2 to 18 hours post-infection (hpi) by vTKhml.
The reaction was then subjected to digestion by RNaseA and RNaseTl under conditions of high salt to inhibit digestion of double-strand RNA. Labeled probc that had hybridized to cellular RNA was subsequently identified following electrophoresis on an 8M urea/polyacrylarnide gel and visualized by autoradiography.
(FIG. 3). A protected RNA fragment of 265 nt which corresponded to transcription of W O 97/13866 PCT~US9G/16368 the insert from the CMV promoter was detected as early as 3 hours post-infection("hpi"), reached high levels by 8 hpi, and m~;nt~incd high levels until 18 hpi. (FIG. 3).
S ISOLATION OFICP4-RECOMBINANTS EXPRESSING THE MlAcHR GENE
Recombinants were generated by homologous recombination between two viruses: dl20, an ICP4(-) virus developed by DeLuca, (DeLuca et al., "Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Immediate Early li~egulatory Protein ICP4," J. Vir. 56:558-570, 1985), and vTE~hml (FIG. lb), prepared in Example 1. Briefly, the viruses were coinfected with E5 cells, an ICP4-expressing Vero cell line. The res~ in~ virus stock was selected for TK(-) mutants with bromodeoxycytidine, and clones were screened for their ability to grow on E5 cells, but not Vero cells.
Positive clones were then tested for the presence of the mlAchR gene by restriction digestion with EcoRI and Southern blot hybr;dization. One virus clone, referred to as vTKhm1-2 (FIG. lc), was found to both express mlAchR and form plaques only with E5 cells.
This recombinant was then used to generate a third recombinant, referred to as vTKhml-3 (FIG. ld), which is defective in both ICP4 and VHS ~:x~ession. E5cells were coinfected with vTKhm1-2 (FIG. lc) and vhsA, the HSV-l mutant that expresses ,B-ga}actosidase from its UL41 region. Bromodeoxycytidine was used to select against vhsA, and the r~sulting viral isolates were screened (a) for their ability to grow on ES cells, but not Vero cells, (b) for the expression of mlAchRs, and (c) for the e~ c;s~ion of ,B-galactosidase. These recombinants were referred to as vTKhm1-3 (FIG. ld).
DETECTION OF SURFACE RECEPTOR EXPRESSION FROM RECOMBINANT
VIRUSES IN VERO CELLS USING LIGAND BINDING ASSAYS
The e2~ ion of mlAchR from Vero cells infected with a multiplicity of infection of 10 with vTKhml-l, vTKhm1-2 and vTKhml-3 was compared using the ~3H]NMS ligand binding assay. Surface mlAchR were measured by incubating 35 infected Vero cells with 1 nM of the radiolabeled muscarinic receptor antagonist, n-methyl-scopolamine ([3H]NMS) at 37~C for 1 hour. After incubation with [3HlNMS, W O 97/13866 PCTrUS96/16368 the infected cells were washed three times with phosphate buffered saline, Iysed and counted in scintillation fiuid. Saturation curves were performed to determine the al,pl~xilllate number of mlAchRs represented by counts measured using lnM
[3H~NMS. (FIG. 4). Competitive inhibition by pi~ ,ille confirmed that these counts reflect specific binding of the ligand to mlAchRs.
Vero cells do not contain any endogenous mlAchRs, therefore any [3H]NMS binding above background represent receptors expressed from the recombinant virus. The expression of mlAchRs from each recombinant is shown.
(FIG. 4). The ICP4-mutant, vTKhrnl-2 infected Vero cells expressed 2-3 fold moremlAchRs 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 those infected with vTKhrn I -1 and at least 2-fold more than 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 t;x~l~,s~ion from vTKhm1-2 and vTKhml-3 are approximately the same. Receptor ex~les~ion from vTKhml plateaus by approximately 12 hpi, and by 36 hpi Vero cells infected with the replication competent vTKhm I -1 recombinant are dead.
DETECTION OF SUR~ACE RECEPTOR EXPRESSION FROM RECOMBINANT VIRUSES
The ~re~ion of mlAchR from E5 cells, ICP4(-~ Vero cells, infected with a multiplicity of infection of 10 with vTKhml-l, vTKhml-2 and vTKhrnl-3 wascompared using the same [3H]NMS ligand binding assay as in Example 4. (FIG. 5).
Complement~tion of the ICP4(-) mutation in vTKhrnl-2 and vTKhrnl-3 transfected E5 cells results in drastically reduced levels of mlAchRs. (FIG. 5). These results indicate that the increased explcs~ion levels in vTKhml-2 and vTKhml-3 infected Vero cells is related to lack of ICP4 ~x~!les~ion. The expression of ICP4 by the E5 cells allows the rccombinant viruses to replicate. (FIG. 11). This data filrther indicates that the lack of viral host-protein synthesis (VHS) exl,lc;ssion contributes to increased mlAchR
expression, since vTKhml-l and vTKhml-3 have higher expression levels than vTKhml-2 in E5 cells.
At 1 hpi and 12 hpi DNA was isolated from each of the infected Vero and E5 cell samples by standard methods and dotted onto nitrocellulose membrane in three fold dilutions. ~FIG. 11) vhsA infected Vero and E5 cell samples served as a control. These results demonstrate that vTKhm1-2 and vTKhml-3 samples replicatedin the E5 cell samples, but not in the Vero cell samples.
Southern blot analysis and immunofluorescence studies were performed to ensure that the recombinants, vTKhml-2 and vTKhml-3, were defective in ICP4 expression. Southern blot confilllled the presence of a 4.05 Icb deletion in ICP4. This deletion is characteristic of dl20, the ICP4(-~ HSV-l strain used to construct these recombinants. The e~ies:iion of the ICP4 product in Vero cells infected with the HSV-1 recombinants was assayed by indirect immunofluorescence using a monoclonal antibody directed against ICP4. Fluolescence micrographs 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 infected with vTKhml-l; vTKhml-2 and vTKhml-3 infected Vero cells did not express cletect~hle amounts of ICP4. (FIG. 9).
EXPRESSION OF MlACHRS FROM HSV-l RECOMBINANTS
IN PRIMARY CORTICAL NEURON CULTURES
Primary cortical neuron cultures, isolated from seven-day-old neonatal rats, were infected with either vTKhml-l, vTKhml-2, or vhsA at a multiple of infection of 3. At 12 hpi, the cultures were in-ub~t~l at 37~C with [3H]NMS for 1 hour. Inaddition, uninfected control cultures were assayed to measure the amount of endogenous mlAchR expressed in primary cortical neuron cultures. Atropine, an mlAchR antagonist which competes with [3H~NMS binding, was used to determine theamount of nonspecific ligand binding present in each sample. (FIG. 7).
In these assays, vTKhml-2 infected cells expressed 5 fold more mlAchRs than uninfected cultures, or approximately 38,000 surface receptors per cell as con~ d to 6,000 receptors on an uninfected cell. (FIG. 6). However, cells infecte~1 with vTKhml-l expressed less than a 2-fold increase in the amount of mlAchR
compared to uninfected cultures. (FIG. 6). vhsA infected cultures ~x~iessed fewer receptors than the uninfected cultures. (FIG. 6). Moreover, there were no cytopathic W O 97/13866 PCTrUS96/16368 effects evident in either vTKhm 1-2 infected neurons or the vTKhml -I infected neurons.
These results demonstrate that the recombinant viruses of the present invention reduce cytopathic effects associated with viral infection and provide heigh1e~t?d ~xl~es~ion of nucleic acid segment inserts.
A phosphatidylinositol turnover assay was performed on neuronal cells infected with each of vTKhml-1, vTKhml-2, and vTKhml -3. This assay demonstratesthat the mlAchR function to stim~ te second messenger systems. 10 d cultures of mouse cortical neurons were infected or mock-infected and then incubated prior to measurement of PI turnover using I uCi/ml [3H] inositol in inositol-free miniln~l 10 essential mcdium. Cultures were washed 3X in Hanks buffered saline solution. Cells were treated or mock-treated with 1 mM carbachol. After 45 rnin, the medium was removed, cells were washed once with HBSS, cold 3% perchloric acid was added, and inositol phosphate levels were determined exactly as described previously (Murphy et al., F~Sl~B J. 4:1624-1633, 1990). Second messengers were st;m~ tçcl S fold by 12 15 hpi in infected Vero cells. Second messengers were stimulated 4 fold in rat cortical neurons.
GENERATION OF A RECOMBINANT HERPESVIRUS VECTOR WHICH
EXPRESSES AN ANTISENSE S-~IT2A RECEPTOR SEQUENCE
A. Materials and Conskuction Methods 1. Vector Construction Plasmid pTKSR2(-) (which is based on pUC 19) consists o~ a Cytomegalovirus (CMV) immediate early promoter, an entire coding region of rat 5-HT2 lecel)lol gene in an ~nti~n~e orientation (see Julius et al., PN~S 87:928-932, 1990; see also Genbank Accession No. M30105), and a polyA sequence from the bovine growth hormone gene. The promoter-~nti~çn~e S-HT2 receptor gene se~uence 30 and poly A region is framed by a Pvu II fragment of the HSV-1 tk gene.
A HSV-1 kos mutant skain vhs A was utilized for construction of recombinant virus. Briefly, in vhs A, a genome region responsible for shutting off host-cell macromolecules synthesis has been deleted. The virus was propagated in African Green Monkey kidney cells (Vero, ATCC Accession No. C~L 1587) cultured with 35 MEM mçdillm with 10% fetal bovine serum (Gibco BRL) at 37~C and 10% CO2.
W O 97113866 PCTrUS96/16368 The recombinant virus was constructed using homologous recombination described above. Briefly, viral DNA from vhs A and pTKSR2(-) plasmid DN~ were cotransferred into Vero cells ntilizing calcium phosphate precipitation. One hundred micrograms of broma-D-oxycytidine (Sigma) was added to the culture to select for tk(-) S virus. Generated virus was collected and diluted with medium to infect Vero cells in 96-well dishes. After 48 hours, medium con~inin~ released virus and cellular debris from the 96 wells were blotted on a membrane followed by a hybridization with a 32p labelled probe made from the 5-HT2 receptor gene. Virus from positive wells werethen plaque purified three times and insertion of the antisense construct was confirmed 10 by Southern blotting and DNA polymerase chain reaction.
2. Preparation of Neuronal Cultures Rat primary cortical cultures were prepared from postnatal day 1 Long-Evans rats. Briefly, each rat was sacrificed by decapitation under halothane anesthesia.
The cerebral cortices were ~ ect~d and minced with iridectomy scissors into fine15 fragments in a brain dissecting buffer solution cont71inin~; Ca2+/Mg2+ free Hank's B~l~nc~e-l Salt Solution (HBSS, Gibco). The fr~gm~ntc were transferred into 3 ml of a solution with 0.16% trypsin (Sigrna) and 0.03% DNase I (Boehrirlger Mannheim) at37~C for 10 minlltes The tissue was then gently dissociated with a 5 ml plastic pipette followed by addition of 2 ml ice cold fetal bovine serum to stop the trypsin activity.
20 The cell suspension was centrifuged and the cell pellets were resuspended in MEM
supplement with 4 mM L-g~ut~min~, 16 mM NaHCO3, 20 mM HEPES and 10% FBS.
After two washes with fresh medium, 2 x 106 cells in 2 ml medium were plated onto poly-L-lysine pretreated glass coverslips in 35 mrn dishes. The culture was incubated in a 5% CO2 incubator at 37~C. The medium was replaced on the following day with fresh 25 medium col.~;~i..;..~ 20 mM cytosine arabinofuranoside (Ara-C, ~igma) to inhibit the proliferation of nonneuronal cells.
3. R~ase Protection Assav Two RNA probes were generated by in vitro transcription. Briefly, probe A was 600 nt with a sequence identical to 5' end sense strain of rat 5-HT2a 30 receptor gene. Probe B was 620 nt in length and spans a region including 300 nt sequence of CMV promoter followed by the first 320 nt sequence of 3' end rat 5-HT2a receptor gene. An RNase protection kit was used and the assay was carried out following the m~nl-f~c*lrer's instruction. The digested hybridized probes were run on 5% Acrylamide gel and exposed on film overnight.
CA 02234604 l998-04-l4 4. Li~and Bindins~Assavs Cells from primary cortical cultures were collected and were freezed-thawed three times before being used for the ligand assays. 106 cells were incubated with 1 nM[3H]ketanserin hydrochloride (85.1 C~i/mmol) or 5nM[3H~mesulergine 5 (Amersham, 750 Ci/mmol) (both from NEN, Du Pont) or 4.5 nMI3H]N-methyl-scopolamine (NMS, 75 Ci/mmol) in Tris buffer (170 mM, pH 7.5) for one hour at room temperature. The non-specific bindings in these assays were less than 20% determined by adding 2,000-fold concentrations of non-isotope-labelled sarne ligands into the correspondin~; incubation solutions. Following the incuh~tion, the cells were washed 10 3 x 5 min in the same buffer at 4~C and re~usl~ellded in 1 ml of ~ormula 963 (NEN) for counting of radioactivity in a Beckman LS 2800 liquid scintillation counter.
B. Results The recombinant virus, vTKSR2(-), co~ ir~g an antisense construct for 15 the rat 5-HT gene was used to infect Vero cells at multiplicity of infection (moi)=3.
Total RNA from the infected cells was collected at 7, 17 and 24 hours postinfection.
Probe A gave rise to a protected band which e~uals to the full length of the probe while probe B was only partially protected and gave rise to a band of 320 nt, the size of transcribed portion. The amount of ~nSi~n~e transcripts increased with the time of 20 postinfection up to 17 hours and declined thereafter.
To prove that the transcribed antisense mRNA could block the ssion of endogenous 5-HT2a receptors in neurons, primary cultures of rat cerebral cortex were used for ligand binding assays following the viral infections. At 7 hours postinfection of vTKSR2(-)(moi=3), the neuronal cultures were double labelled with a 25 monoclonal antibody against microtubule-associated protein type 2 (MAP-2), a neuron-specific marker, and a polyclonal antibody against HSV-l virus. More than 90% ofcells in the cultures were labelled by MAP-2 antibody indicating their neuronal nature.
Among the MAP-2 positive cells, more than 80% of them were also showed immunoreactivity of HSV-1 antigens, indicating that a large population of the neurons 30 were infected by the recombinant virus. Following the infection with vTKSR2(-) or the parental virus vhs A at moi=3, neurons were incubated with [3H3ketanserin or [3H]NMS
at either 7 hours, 17 hours and 48 hours postinfection. The radioactivity of bound [3H]ketanserin was significantly reduced in cultures infected with vTKSR2(-) comp~rin~ to the VHS A infected ones. The reduction of binding in neuronal cultures 35 peaked at 17 hours and declined in 48 hours postin~ection. In contrast, there was no difference in binding of [3H]NMS between the vTKSR2(-) and VHS A infected W O 97/13866 PCTfUS96/16368 cultures. Furthermore, using ~3H3mesulergine, a ligand more specific for 5-HT2c receptors, to label the infected cultures, a minor reduction of binding was observed with a marginal significance of statistics.
IN VIVO ADMIN~STRATlON
Long-Evans rats were utilized as subjects to determine the degree of infectivity of recombinant Herpesvirus vectors. Briefly, the rats were ~n~ctheti~cl with an intraperitoneal injection of sodium pentobarbitol (Somnotol, 40 mg/kg), and placed into a stereotaxic appal~Lus. A craniotomy was performed over the appropriate brain structure, and the Herpesvirus vector SHT2 described above in Example 8 was injected.
In the case of one rat, a total of 10 ul at a titer o~ 108 pfu/ml was injected into the hippocampus over a period of 20 mimlt~ The rat was then sutured and treated with a topical antibiotic (chloramphenicol, 1%). After 4 days, the rat was sacrificed and the hippocampal region sectioned and stained with an immunohistochemical to recognize the viral envelope. The results are shown in Figure 12B. Briefly, infection can be seen in a substantial number of cells within the needle track.
Another rat was injected in the cortex with 200 ul at a titer of 5 x 107 pfulml over a period of 90 m;n~ltes As above, the rat was then sutured and treated with a topical antibiotic. After 2 days the rat was sacrificed and the cortex sectioned and stained. Results are shown in Figure 12A. Briefly, strong labelling (and thus, infection of cells) can be seen throughout the periphery of the cortex.
CONSTRUCTION AND ASSAY OF NEURON-SPECIFIC VECTORS
A. M~tçri~l~ and Construction Methods All restriction endonucleases, media, fetal bovine serum (FBS), nerve growth factor (NGF) and fine chemicals were obtained from GIBCO BRL Life Technologies Inc. (Burlington, Ontario~. Unless otherwise noted, cloning procedures followed standard procedures (Sambrook etal., "Molecular Cloning: A laboratory m~nu~l," 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
Plasmid pON134 contains the HSV-l (strain KOS~ latency-associated transcript promoter (LAPl) (Selden etal., Mol. Cell. Biol. 6:3173-3179, 1986; Ho and W O 97tl3866 PCT~US96tl6368 Mocarski, Proc. Nal'l Acad. Sci US~ 86:7596-7600, 1989). Briefly, the LAPl promoter is located immediately u~ t;;anl of the large primary approximately 8.5kb LAT RNA transcript and about 650 bp U~iL~ of the 5' ends of the small LAT RNAs.
As shown in Figure 13, this promoter COllt~ S a typical TATA box and several 5 characteristic eukaryotic transcription regulatory elements, such a cyclic AMP-response elements, a CATT box, and two Spl sites. One region of the LAP promoter (betweenabout -161 and about -259~ is particularly preferred for conferring a neuronal cell-type specific activity.
The LAP1 promoter was cloned from pON134 by digestion of the 10 plasmid with PvuI and a 609 bp DNA fragment containing the LAP l promoter from ~2 to -608 (Batchelor and O'Hare, ~ Yirol. 66:3573-3582, 1992; McGeoch et al., J. Gen.
ViroL 69:1531-1574, 1988) was inserted at the Hinc II site of the pBluescript KS (+) vector (Stratagene, LaJolla, Califorr~ia). The minimum LAPl promoter used in ~}is study was constructed by a filrther SstII deletion of the upstrearn sequences at -259.
Dimer neuronal restrictive silencer elements were constructed by ~nne~lin~ two synthetic oligonucleotides essentially as described by Mori et al. (Neuron 9:45-54, 1992), S36~: CAAAGCCATT TCAGCACCAC GGAGAGTGCC TCTGC
(Sequence 1.I~. No. 2) and S36B: GCAGAGGCAC TCTCCGTGGT GCTGAAATGG
CTTTG (Sequence I.D. No. 3), followed by ligating the double-stranded DNA
2û fragments and inserting them into a pBluescript KS(+) vector at the Hinc II site. The correct sequences and orientations of dimer silencers were examined and verif1ed by standard double-stranded dideoxynucleotide sequencing.
The human cytomegalovirus (hCMV) major immediate early gene enhancer from -113 to -601 (Boshart etal., Cell 41:521-530, 1985) was cloned from 25 pRc/CMV (lnvitrogen, San Diego, California) by digestion of the hCMV major immediate early promoter with Ban I and Hinc II so that the TATA box of the promoter was deleted. The 488 bp enhancer fragment was inserted at the Hinc II site of pBluescript KS(+).
A 21kb BamHI-EcoRI human growth hormone (hGH) gene fragment 30 including its own polyadenylation signal was cloned out from a plasmid pOGH (Selden et al., Mol. Cell. Biol. 6:3173-3179, 1986).
B. Cells. Transfections and hGH Assay Vero and rat pheochromocytoma (PC12) cells were obtained from the 35 ATCC. Briefly, Vero cells are monkey kidney cells which are capable of supporting HSV-I propagation in vitro. PC12 cells are neural crest derived rat W O 97/13866 PCTrUS96116368 phaeochromocytoma cell line. In response to nerve growth factor, PC12 cells stopdividing and differentiate into neuron-like cells with many properties characteristic of peripheral nervous tissue.
Vero cells were propagated in Oulbecco's Modified Eagle Medium 5 (DMEM) with 10% FBS and 10% inactive horse serum. Difrele,lliation of PC12 cells induced by NGF at a concentration of 150 ng/ml was performed at 16 hours post-transfection. Transfection of Vero and PC12 cells with the plasmid constructs on six-well dishes with 3 x 105 cells per well was performed lltili7inl7 the calcium phosphate-me~ t.ocl precipitation method essentially as described by Sambrook et al., supra. The 10 amount of DNA used in the transfection was titrated to 5 ug per well. The linear response range of hGH accllm~ cl in the medium was det~rm;ned hetween 48 to 96 hours after transfection for both cell types and therefore, the media were collected at 48, 72 and 96 hours after transfection. The amount of hGH secreted into the medium was measured with a solid-phase two-site radioimmunoassay Icit under the conditions 15 recommen-le~l by the m~nllf~c$ure (Nichols Tn~tit~lt~ Diagnostics, Los Angeles, California). Data were collected from at least two sets of transfection experiments and each transfection was performed in triplicates.
C. Chimeric ~ ei,~ion cassettes with LAPl promoters and NRSE silencer 20 elements To selectively alter LAP 1 promoter activity only in neuronal cells, several synthetic NRSE dimers were placed upstream of the LAPl promoter in all possible orientations (Figure 14A) and tested in both Vero and PC12 cells by transient expression assays. As shown in Figure 14~3, the NRSE monomer (pSA/LAP/HGH) 25 su~lc;ssed the LAPl promoter activity by only 20% in Vero cells and had no effect on the promoter activity in PC12 cells as co~ al~d to the LAPl promoter without NRSE
(pLAP/HGH). Moreover, when the chimeric LAPl promoters with the head-to-tail NRSE dimers in either orientation (pS2A/LAP/HGH and pS2B/LAP/HGH) were introduced into Vero cells, the promoter activity decreased to 60 to 80% relative to the 30 parental plasmid construct (pLAP/HGH). These NRSE dimers however, did not inhibit LAPl promoter activity in PC12 cells. In addition, chimeric LAP1 promoters with either head-to-head or tail-to-tail NRSE dimers (pS2H/LAP/HGH and pS2T/LAP/HGH) showed somewhat lower promoter activity (about 60%) as compared to the construct without NRSE and had no cell-type p.~felence for Vero or PC12 cells.
35 From these results it was concluded that an NRSE element efficiently suppressed the HSV-1 LAP1 promoter in non-neuronal cells and an NRSE dimer was more effective than a monomer when placed in a head-to-tail orientation. Therefore, the chimeric promoter construct pS2B/LAP/HGH was chosen for further experiments.
The hCMV enhsm~çr element from -113 to -601 of the hCMV major IE
promoter was placed either u~ c~ll of the LAP 1 promoter (pE/LAP/HGH) or at the 3' S end of the hGH reporter gene (pLAP/HGH/E). The resulting chimeric promoter constructs were introduced into both Vero and PC12 cells. As shown in Figure 15B, the LAP1 promoter activity was stimulated by the enhancer in both cell types as compared to the basal level activity of the basic LAP I promoter construct pLAP/HGH in each cell line, which indicates that the enh~n~ ~r element worked in both cell types. Moreover, in 10 Vero cells, the LAP I promoter activity was up-regulated by about 2.5 fold regardless of the position of the enhancer. By contrast, in PC12 cells, when the hCMV enhz~nl~er was placed at the 3' end of the hGH gene (pLAP/HG~/E), the enh~n-er activity was five times higher than when the enhancer was upstrearn of the LAP l promoter (pE/LAP/HGH).
To evaluate the potential cell-type l~ler~r~llce contributed by different promoters, the hCMV major immediate early (IE) promoter with its own enh~nt~er was included as a control. As shown in Figure l5B, in Vero cells the hCMV major IE
promoter itself had a much higher activity (fourteen fold) than the LAP1 promoter, whereas in PC12 cells it had the same activity as the LAP1 promoter. These results suggest that the relative hCMV enhancer activity in dirr~lclll cell types was well dependent on the promoter to which it was linked. In addition, since the enh~n~relement itself showed no promoter activity in either Vero or PC12 cells, the chimeric LAP1 promoter must have been enh~nce~ as a result of interactions between the LAP1 promoter and the hCMV enh~ncer element. These results demon~tr~ted that the HSV-1 LAP1 promoter could be up-regulated in both neuronal and non-neuronal cells by an enhancer from the hCMV major IE promoter. However, the level of enhancement of the LAPI promoter in neuronal cells was dependent on the position of the enh:lnr~r element relative to the promoter.
In summ~ry, the HSV-1 LAP1 promoter can be regulated by other gene regulatory sequences either to ~7U~lC3s the promoter activity in non-neuronal cells, or to enhance its activity in both neuronal and non-neuronal cells.
D. Chimeric e~ es~,ion cassettes with LAP I promoters. NRSE silencer elements and hCMV enhancers The chimeric LAP1 promoter construct cont~inin~ the silencer dimer, pS2B/LAP/HGH, was used as a backbone for further studies. Briefly, this construct W O 97~13866 PCT~US96/16368 contained a head-to-tail NRSE dimer upstream of the LAP1 promoter and had the lowest promoter activity among the constructs tested in Vero cells (Fig. 14B). The hCMV enhancer element was added either ~ l of the LAP 1 promoter (pS2B/E/LAP/HGH), or at the 3' end of the hGH reporter gene (pS2B/LAP/HGH/E), as~ 5 shown in Fig. 16A. When pS2B/E/LAP/HGH and pS2B/LAP/HGH/E constructs were introduced into Vero and PC12 cells, they all showed a higher level of promoter activity in both cell types than the construct pS2B/LAP/~GH, which lacks the enh~ncer element. Moreover, in Vero cells, the NRSE dimer no longer ~ essed the LAP1 promoter activity in the presence of the enhancer (lF ig. 1 6B). Thus, the hCMV enh~nc.er 10 activity was dominant over the silencing activity of the NRS~ dimer element when they were combined. Interestingly, the enhancer also showed position effects on the LAPl promoter activity in the presence of the NR~E dimer. When it was placed at the 3' end of the hGH reporter gene (pS2B/LAP/HGH/E), it showed a stronger ~nh~nfçment of the LAPl promoter in PC12 cells than in Vero cells as demonstrated previously (~ig. lSB).
15 By contrast, when it was placed at the 5' end of the LAP 1 promoter (pS2B/E/LAP/H~H), it showed an unusually high level of activity in Vero cells, whereas in PC12 cells it showed only slight enhancing activity.
~. The Chimeric LAPI Promoter Activity in the Differentiated PC12 Cells 20 Tn~ cecl bYNGF
Nerve growth factor (NGF) is important for the m~ te.~sln~e of neuronal cell survival. In the presence of NGF, PC12 cells undergo various physiological changes and differentiate into neuron-like cells with many characteristics of peripheral nervous tissue.
In order to test the responses of LAPl promoters in NGF in~ e-1 PC12 cells, chimeric LAPl promoter constructs were first transfected into PC12 cells and then cell ~liLrt;~ iation was incln~ed by treatment with NGF. As shown in Fig. 17B, the LAP1 promoter activity increased about four fold in response to NGF, whereas the chimeric LAPl promoter with the hCMV enh~n~er element (pLAP/HGH/E) showed 30 two fold increased activity. Interestingly, the hCMV major IE promoter also increased its activity to the sarne extent as the LAPl promoter in response to NGF tre~tment in ~ PC12 cells, which suggests that the up-regulation of the LAP1 promoter in the differenti~ted PC12 cells was part of an overall stim~ tion of transcription in response ~ to NGF.
From the foregoing, it will be evident that although specific embo~imenf~ of the invention have been described herein for the purposes of W O 97/13866 PCT~US96/16368 illustration, various modifications may be made without deviating from the spirit and scope of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Cynader, Max Tufaro, Franci s (ii) TITLE OF INVENTION: METHOD OF USING, PROCESS OF PREPARING.
AND COMPOSITION COMPRISING RECOMBINANT HERPESVIRUS VECTORS
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(B) FILING DATE: 11-OCT-199 (C) CLASSIFICATION:
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(A) NAME: McMasters, David D.
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(A) TELEPHONE: (206) 622-4900 (B) TELEFAX: (206) 682-6031 CA 02234604 l998-04-l4 (2) INFORMATION FOR SEQ ID NO:1:
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(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
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GCTGAAATGG C m G 15
G PRoTElN-LINKED RECEPTORS AND RECOMBINANT HE~PESVIRUS VECTORS
Within the various aspects of the present invention, the expression c~settes or gene delivery vehicles described herein (e.g, recombinant Herpesvirus vectors may be utilized as a means of introducing nucleic acid segments into nonrnitotic cells primarily of the nervous system (collectively referred to as "neural" or "neuronal"
cells). For example, recombinant He,l,e~vi,us vectors of the present invention may be utilized to deliver nucleic acid segm~nt~ into the cell where the proteins are expressed, generally as mRNA which is then tr~n~l~te~l into a protein. When the protein tr~n~l~tecl 3~) is a G protein linked receptor, for example, the 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 me~nger system and, thus, function in much the same manner as a naturally occurring receptor.
- Briefly, Herpesviruses such as HSV-I are double stranded DNA viruses 35 (approx. 152 kb) which are replicated and transcribed in the nucleus of the cell.
Although HSV-l is utilized as a representative Herpesvirus in the description and examples provided below, it should be understood that the present invention is not so limited. In particular, numerous other Herpesviruses (including for exarnple~ those described above), may be utilized within the context of the present invention.
Productive infection by HSV-l usually results in cell Iysis or alteration 5 of host macromolecular processes. However, HSV~1 also may be m~int~ined in~lefinitely in the "latent state" in certain cells by a mechanism involving the tegument of the virus particles. The reactivation of the virus is regulated by certain systemic or cellular events. The latent virus is still transcriptionally active, producing "latency associated transcripts" (LATS). Mutant viruses that are compromised or defective in 10 their replication potential can still enter the latent state (e.g., UL41(-), TK(-), and ICP4(-)). In fact, a TK(-) HSV-I will mslint~in the latent state in~efnitely. Thus, HSV-I is ideal for use in delivering nucleic acid segments to non-mitotic cells such as neuronal cells. Within the present invention, Herpesvirus vectors such as those derived from HSV-I are preferably m~mt~inP!d in the latent state.
The manipulation of Herpesviruses such as ~ISV-I for the purposes of the present invention, may, within certain aspects involve deletions, substitutions or mutations of non.?~c~nti~l regions of a Herpesvirus genome, generally m;.;~L~ir~i"~ the essential regions intact. In the context of the present invention, "essenti~l region" refers to any region of the viral genome the deletion of which would result in an inability to 20 infect a m~mm~ n host cell or an inability to replicate, even with the assistance of a helper virus or a complementing cell line. Non~?sct?nti~l 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 re~uired for 25 the propagation of replication defective viruses that can not, by defînition, propagate themselves. Such helper viruses are described in Fields et al., Fundamental Virology, Raven Press, N.Y. (1986), and are well known to those skilled in the art. Examples of helper viruses suitable for use in the present invention include unaltered Herpesviruses such as HSV-l as well as other viruses that express the genes contained within the 30 deleted region whose products are nec~ ry for propagation of a recombinant Herpesvirus.
The terrn "complementin~ cell lines" refers to cell lines that provide gene products re~uired for the propagation of defective viruses that by definition cannot propagate themselves. Suitable complem~ntin~ cell lines in the present invention35 include E5 Vero cells, which provide the protein ICP4 for replication deficient viruses.
(Disclosed in detail in DeLuca et al., J. Yir. 56:558-570, 1985.) CA 02234604 l998-04-l4 W O 97~13866 PCT~US96/I6368 As noted above, within certain aspects of the present invention, nucleic acid segments are inserted into a Herpesvirus genome and/or portions of the Herpesvirus genome are deleted. Preferably, insertions or deletions of nucleic acid segments utilized in the present invention are made to one or more of the following 5 nonessential regions: the UL41, thymidine kinase (TK), and/or any one of several replication loci (Ward and Roizman, Trends in Gen 10:267, 1994). The replication loci include DNA polymerase and that for the ICP4 protein. Briefly, ~CP4 is a proteinproduced by a viral immediate-early gene and governs transcriptional regulators required for the e2.~lc~ion of the early genes. Likewise, Thymidine kinase is an early 10 gene implicated in the replication of viral DNA. UL41 is a late gene whose protein product is responsible for early shut off of host cell macromolecular synthesis.The Herpesvirus genome can be manipulated to produce such deletions and insertions by using standard recombinant DNA techniques, such as those described in Maniatis etal., Molecular Cloning, ~ Laboratory Manual, Cold Spring Harbor 15 Laboratory, Cold Spring Harbor, N.~. (1982), or Sambrook et al., Molecular Cloning, A Laboratory Manu~l, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). Briefly, deletions within a Herpesvirus genome can be effected by conventional techniques employing endonucleases, exonucleases and the like. Insertions can also be executed using conventional techniques, including, by way 20 of example cotransfection, i.e., homologous recombination facilitated by a suitable plasmid. A suitable plasmid available for such use includes pl~c/CMV (Invikogen Corp.). The plasmid incll-r1ing the desired characteristics can be selected using conventional methods and introduced for propagation purposes into a host cell ororganism using standard transformation procedures. The plasmid is then isolated from 25 the host org~ni~m, mixed with unaltered Herpesvirus DNA and cotrzln~fecte~ into host cells. The cells cont~inin~ the plasmid and the Herpesvirus DNA are cultured, and homologous recombination take place, resulting in the replacement of the unaltered region in the viral DNA with the corresponding altered region from the plasmid. Any host cell suitable for plasmid and ~erpesvirus DNA transfection and subsequent 30 recombinant virus propagation can be utilized in this procedure. The recombinant Herpesvirus DNA is then replicated within the cell and the viruses which have undergone the desired recombination are selected using standard techniques.
As noted above, recombinant Herpesvirus of the present invention are produced through insertion of nucleic acid segments into the genome. Within the 35 context of the present invention, "nucleic acid segment" refers to a nucleic acid sequence or molecule, and may be derived from a variety of sources including DNA, CA 02234604 l998-04-l4 W O 97/13866 PCT~US96/16368 cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid segments may comprise genomic DNA which may or may not include naturally occurring introns.
Such genomic DNA may be obtained in association with promoter regions or poly A
se~uences. Further, The nucleic acid segment may be aIl antisense sequence. The 5 nucleic acid segments of t~e 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 any one of several means. Alternatively, mRNA can be isolated from a cell and used to produce cDNA ~oy reverse transcriptase by any one of several methods.
Within particular prefe~red embo-~iment~ of the present invention, the nucleic acid se~ment is a G protein linked receptor gene. In the context of the present invention, the term "G protein linked receptor" refers to a gua~ine nucleotide binding regulatory protein coupled to both a cell surface receptor and an effector, such as an ion channel, together comprising a tr~n~memhrane .~i~n~lin~ system. G protein linked15 receptors me~ t~ the actions of extracellular signals, such as neurotr~n.~mitt~rs. They are described in detail in Dohlman etal., Ann. Rev. Bioc*em. 60:553-588 (1991~.
Suitable G protein linked receptors genes include those listed in Table I and portions thereof.
It will be evident to those skilled in the art that the particular receptor 20 utilized will be influenced by the characterist;cs of the receptor and the specific tre~tm~nt Receptor Subtype Species Ref.
M~m~
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Although it is preferable to utilize the complete coding sequence from the G protein liinked receptor gene, within certain embodiments of the invention only that portion of the G protein linked receptor gene which encodes expression of the 5 receptor on the cell surface need be utili7~o~1 Within the context of the present invention, both the entire coding region and portions thereof are referred to as "G
protein linked receptor genes." Such ex~ ion can be ~letermin~l by any one of several suitable means, including ligand binding assays.
The coding sequence for the G protein linked receptor should be inserted in such a manner that the resulting recombinant Herpesvirus genome directs the synthesis of a L~ sc.ipt that is capable of being trRn~ t( ~1 into a G protein linked receptor protein. The desired G protein linked receptor produced should be compatible S with Herpesvirus propagation (i.e., is not lethal). The promoter sequence can be supplied within a separate or the same nucleic acid segm~nt as the G protein linked receptor sequence or by the genomic portion of the recombinant virus. Suitable promoters include any one of several which are capable of initiating expression of the G
protein receptor gene. Preferably, the promoter is a major immediate early promoter 10 and the sequence includes a polyadenylation site. More preferably, the promoter is the cy~omegalovirus (CMV) promoter.
In a preferred embodiment of the present invention, the Herpesvirus utilized is deficient for ~ t;ssion of the thymidine kinase (TK) gene locus (TK(-)).
More preferably, the G protein linked receptor sequence is inserted in the thymidine 15 kinase (TK) gene locus of an HSV-l genome, rendering it deficient. Within the context of the present invention, "deficient" refers to low or nonexistent expression of the gene in question. Deficient expression generally results from insertion into or deletion of the genet~c loci in question. Deficiency of the thymidine kinase loci can be assayed using any one of several means, including selection with bromodeoxyctidine using standard 20 methods (see also Surnmers et al., PNAS 72:4081, 1975).
In another preferred embodiment of the present invention, the Herpesvirus genome ;s deficient for the expression of virion host shut off gene (IJL4 1 ) locus and the thymidine kinase (TK) gene locus. Even more preferably, a nucleic acid segment encoding beta-galacto~ e is inserted in the virion host shut-off gene (UL41) 25 locus to allow for easy confirmzltion of successful debilitation and the G protein linked receptor sequence is inserted in the thymidine kinase (TK) gene locus. The deficiency in UL41 ~ression may be assayed for by ~letecting beta-galactositl~e expression using standard techniques (see also Smibert et al., J. Vir. 68:2339, 1994).
In another aspect of the present invention, the Herpesvirus genome is 30 a~ditionally deficient in the expression of a viral gene required for replication ("replication deficient"). Briefly, proteins required for replication include, by way of example, ICP4 and DNA polymerase. Preferably, it is replication deficient in thes~ion of the ICP4 protein. Replication deficiency can be assayed using any one of several standard methods, including by comparison of cultures in complementary and 35 noncomplementary cell lines (see also DeLuca et al., J. Vir. 56:558-570, 1985).
wc~ s7rl3s66 PCT~US96~16368 In another embodiment of the present invention, HSV-l 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 UL4 1 loci and ICP4 protein.
Within the context of the present invention, "vTKhml-l" refers to a recombinant Herpesvirus vector which is deficient in both the expression of the viral host shut offprotein (VHS) and thymidine kinase (TK). (FIG. lb); "vTKhml-2" refers to a recombinant Herpesvirus vector which is deficient in the expression of both the viral transcriptional regulator (ICP4) and thymidine kinase (TK). (FIG. lc); and"vTKhml-3" refers to a recombinant Herpesvirus vector which is deficient in both the e~x~le~SiOn of the viral transcriptional regulator, ICP4, VHS, and thymidine kinase (TK). (FIG. ld). All three of the recombinant viruses express a G protein linkedreceptor (preferably inserted in the TK locus) from an imm~ te early promoter, preferably a CMV promoter. As described in more detail below, these recombinant Herpesvirus vectors are characterized by low cytopathicity and a high rate of expression. Recombinant Herpesvirus vectors with "çssenti~lly the same ch~r~cteristics"
is intended to refer to recombinant Herpesvirus vectors with the same or similardeficiencies in expression.
These and other recombinant Herpesvirus vectors characterized by low cytopathicity and/or a high level of ~ ion of G protein linked receptor may be produced by cllltllring a first and second recombinant Herpesvirus in a suita~le cell line for a time sufficient and under suitable conditions to allow for recombination. The first recombinant Herpesvirus is one carrying a G protein linked receptor gene and capable of expression thereof and the second recombinant Herpesvirus is replication deficient.
The G protein linked receptor nucleic acid segment may be inserted into the first recombinant Herpesvirus by any suitable means described above, including homologous recombination between the virus and a plasmid carrying the G protein linked receptor nucleic acid segment Recombinant Herpesvirus vectors carrying the G
protein linked receptor sequence may then be selected for using standard methods, including restriction digestion followed by Southern Blot hybridization. Preferably, the first recombinant Herpesvirus is TK(-) HSV-l. Even more preferably, the G protein linked receptor gene is inserted in the TK locus of the first recombinant Herpesvirus.
Additionally, the first recombinant Herpesvirus is preferably deficient in expression in the virion host shut-off protein (VHS). Most preferably, the first recombinant virus is vhsA (J. Smiley, McMaster U~livelsiLy, Hamilton Ontario) (FIG. lA). Briefly, vhsA is a mutant HSV-l which bears the beta-galactosidase gene in the UL41 region of its CA 02234604 l998-04-l4 W O 97/13866 PCT~US96/16368 genome, rendering it deficient in expression of the virion host shut-off protein. The G
protein linked receptor gene may be inserted into vhsA by the means described above.
Preferably, the second recombinant Herpesvirus vector is replication deficient. Even more preferably the second recombinant Herpesvirus vector is def1cient in the expression of the ICP4 protein. Most preferably, the second recombinant HSV-1 is dl20. (Disclosed in detail in DeLuca etal., "Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Irnmediate ~arly Regulatory Protein ICP4," ~ Vir. 56:558-570, 1985). Briefly, dl2Q is replicationdeficient HSV-l, due to 11iminisht-d ~ ession of ICP4. Recombinants defective for 10 ICP4 expression may be selected using any one of several suitable methods noted above including Southern blot analysis, Northern blot analysis, or immunofluorescence studies.
If both the first and the second recombinant Herpesviruses are replication deficient, the two recombinant Herpesviruses can be transfected on a complementary 15 cell line for replication. Suitable complement~ry cell lines include E5 Vero cells (ICP4(+)). (Disclosed in detail in DeLuca etal., "Isolation and Chara~ ion ofDeletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Immediate EarlyRegulatory Protein ICP4," J. Vir. 56:558-570, 1985).
The recombinant Herpesvirus vector resulting from the transfection of 20 the first and second recombinant Herpesviruses may be selected for one or more of four basic characteristics: (1) thymidine kinase deficiency, (2) ICP4 expression, (3) UL41 ~xp~e~ion, and (4) G protein receptor gene ~p.~,s,ion, using any one of several suitable methods described above. By way of example, thymidine kinase expressioncan be screened for using bromodeoxycytidine; ICP4 expression can be screened for :25 based on the virus' ability or inability to grow on the complementin~ cell lines; UL41 expression can be screened for based on beta-g~ rt~idase production, and t;~lt;ssion of the G protein linked receptor gene can be screened for based a on ribonuclease protection assay. Maniatis etal., Molecular Cloning, ~ Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Thus, three ple~-~t;d30 embodiments of the invention vTKhml-l (FIG. lb~, vTKhml-2 (FIG. lc), and vTKhml-3 (FIG. ld) may be produced and screened a~cording to ~2~ple~ion. The more preferred embodiment is vTKhml-3 (FIG. ld).
As noted above, within other aspects of the present invention, recombinant Herpesvirus vectors can be used to deliver G protein linked receptor~5 nucleic acid sequence to mslrnm~ n cells. Once infected, the recombinant Herpesvirus vector will then produce the desired receptors which are expressed on the cell surface.
, WO g7/13866 PCT~US9Cf~6368 The infected cells are then selected for the desired G protein linked receptor expression.
For virus infection, the recombinant Herpesvirus vectors may be applied to the cells under standard cell culture conditions. Cell culture techniques are described in Maniatis et al., Molecular Cloning, ~4 Laboratory Manual, Cold Spring Harbor Laboratory, Cold 5 Spring Harbor, N.Y. (1982~. The specific host cells employed in the present invention are not critical as long as they allow replication and ~pl~s~ion of the recombinant Herpesvirus vectors. Suitable cells include Vero cells (ATCC Accession No. CRL
1587).
To select for the t;~ t;s~ion of G protein linked receptors, standard 10 techniques may be employed, including ribonuclease protection assays such as those described in Maniatis et al., Molecular Cloning, ,4 Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Briefly, a labeled RNA probe is synthesi7~-1 that is partially complemcnt~ry to the region of the target mRNA. This labeled RNA probe is added to samples of the total cellular RNAs isolated from the cell 15 culture after post infection by the recombinant virus. The mixture is incub~te~l, for a sufficient time and under suitable conditions to enable a labeled probe to hybridize to the complementary RNAs and then subjected to digestion by suitable restriction enzymes, such as RNase A and RNase Tl. Labeled probe that hybridized to complementary transcripts will be protected from digestion and may be separated on a 20 polyacrylamide gel and viewed by autoradiography.
G linked protein receptor e~ ion resulting from the insertion of the recombinant viruses of the instant invention into the cell can be detected using any one of several methods known in the art, including for example, ligand binding assays.
Representative ligand binding assays suitable for use within the present invention 25 include those described in Conn, Methods in Neurosciences (Vol. 9), "Gene Expression in Neural Tissues" Academic Press, Inc., San Diego, ~alifornia (1992). For example, within one embodiment the cells infected with the recombinant virus are incubated with a radiolabeled antagonist. Saturation curves may then be performed in order to determine the approximate number of receptors ~represented by counts measured using 30 the antagonist and competitive inhibition). Within other embo-limentc, stimulation of second messenger systems maybe be ascertained by any one of several suitable means, including, for example, phosphatidylinositol (PI) turnover assays.
The recombinant viruses of the present invention may be characterized in a variety of manners, including for example, by the number of receptors expressed on 35 cells infected with the virus, the in vivo cytopathicity of the virus, and/or the immlln~genici~y of the virus. For example, within certain embodiments of the present CA 02234604 l998-04-l4 W O97/13866 PCT~US96/16368 invention, recombinant Herpesvirus vectors are provided which express greater than about 10,000 receptors per cell, preferably between about 25,000-200,000 receptors per cell, more pre~erably greater than about 200,000 receptors per cell and n1ost preferably between about 200,000 to 400,000 receptors per cell, or even greater than about 400,000 5 receptors per cell. Within other embodiments, recombinant viruses are provided which have an in vivo cytopathicity of general}y less than the in vitro cytopathicity."Cytopathicity" as used herein, refers to cell survival five days after infection.
Cytopathicity may be measured using any one of a wide variety of techniques known in the art, including commercially available kits. Suitable kits include Live/DeadTM
10 (Molecular Probes Inc.; viability/cytotoxicity kit lltili7ill~ a method of staining).
By way of example, vTKhml-1 is characterized by a surface receptor expression rate generally greater than 10,000 receptors per cell; typically in about the range of 60,000 to 80,000; and preferably in about the range of 70,000 to 100,000.
vTKhml-2 is characterized by a cytopathicity of generally less than 15 50%; typically in about the range of 35%-40%; and preferably in about the range of 20%-35%. vTKhm1-2 is further characterized by surface receptor expression generally greater than 80,000; typically in about the range of 120,000 - 160,000; and preferably in about the range of 160,000 to 200,000.
vTKhm1-3 is characterized by a cytopathicity of about in vi~ro ~0 cytopathicity of less than about 3%, typically in about the range of 0.1% to 1.0% aIld preferably in about the range of .001% - 0.1%. vTKhm1-3 is further characterized by surface receptor expression generally greater than 800,000 receptors, typically in the range of 1-1.5 million receptors, and preferably 1.25-2 million receptors/cell.
ADDITIONAL METHODS AND COMPOSITIONS
Within other aspects of the invention, methods are provided for producing a protein of interest, comprising the steps of introducing an t;~ e~ion cassette or gene delivery construct as described above, into a host cell, and culturing the hot cell under conditions, and for a time sufficient to permit ~xplc~ion of the protein.
3~) The expression cassettes may be introduced by a wide variety of mech~ni~m~, including for example, including for example calcium phosphate-mediated transfection (Wigler et al., C~ 4:725, 1978), lipofection; gene gun (Corsaro and Pearson, Somatic Cell Gen.
7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (NeumRnn et al., EMBO J. I:841-845, 1982), protoplast fusion-mediated transfection or DE~QE-dextran me~liRt~d transfection (Ausubel et al., (eds.), Current Protocols in Molecu~ar Biology, John Wiley and Sons, Inc., NY, NY, 1987).
=
WO 97~3866 PCT~US96~16368 Optionally, the resultant protein may be purified by a variety of methods, including for example, within one embodiment cell super~t~nt may be first concentrated using cominercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following concentration, the 5 concentrate may be applied to a suitable purification matrix, or ~Itern~tively, purified utilizied anion or cation çxeh~n~e resins. Finally, one or more reversed-phase high olll.ance liquid chromatography (RP-HPLC) steps may be employed.
Expression cassettes or gene delivery constructs of the present invention may also be utilized to introduce a selected sequence of interest into an in vitro culture 10 cont~ininP neuronal cells, comprising the step of introducing an expression cassette or gene delivery construct, as described herein, into an in vitro culture of cells cont~ining neuronal cells.
In another aspect of the present invention, and using the techniques described above, the gene delivery constructs or recombinant viruses of the present 15 invention can also be packaged in a suitable cell line. For example, within one embodiment of the invention, recombinant HSV-I is cultured ex vivo in suitable m~mmz~ n cells. These cells may then be introduced in vivo, using the techniquesdescribe below, i.e., stereotactical microinjection, for l~e~l...cnt of neurological disorders or analysis. Alternatively, the t:x,~les~ion cassette or gene delivery construct 20 may be introduced directly in vivo by any one of several methods described below.
In another aspect of the present invention, the ~x~-~s~ion cassettes or gene delivery constructs described above are a-lminict~red to a warm-blooded animal (e.g, a human, monkey, cow, sheep, dog, cat, rat or mouse), or other-type of animal (e.g, fish) for the tre~trnent of neuronal cell disorders, in both the central and peripheral 25 nervous system. Such c~ett~s or constructs may be utilized in the tre~tment of a wide variety of disorders, including for example, brain turnors, degenerative disorders, neural disorders characterized by abnormal gene expression, and inherited disorders.
The ~ s~ion cassettes or gene delivery constructs of the present invention may also be utilized to deliver normal genes. This allows for the tre~tment of 30 deficiency state disorders, usually of enzymes, by increasing production thereof.
Additionally, the recombinant 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 ~ s~iv~ disorders. For example, expression c~ettes of the present invention may be utilized to express sequences of interest in non-human 35 transgenic ~nim~ such as mice, rats, rabbits, sheep, dogs and pigs (see Hammer et al.
(Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), Brinster et al.
W O 97/13866 PCTrUS96tl6368 (Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985), Palmiter and 13rinster (Cell 41:343-345, 1985) and U.S. Patent No. 4,736,866). For example, within one embodiment anexpression c~sette may be introduced into pronuclei of fertilized eggs, for example, by microinjection. Integration of the injected DNA may be detected by blot analysis of S DNA from tissue samples. It is pl~rcl.~d that the introduced DNA be incorporated into the germ line of the animal so that it is passed on to the animal's progeny. Such techniques allow for, within preferred embodiments, tissue-specific (eg., neuronai cell) ession of a desired sequence of interest.
The ~x~-~s~ion c~ette~ or gene delivery constructs of the present 10 invention may also be used to create nnh~l~nced state disorders involving structural or regulatory proteins, in a model system, which could be used in efforts to establish and study methods of counteracting the effect of the imh~ nce.
In one aspect of the present invention, ~x~iei,~ion r~settes or gene delivery constructs may be used to treat neurodegenerative disorders including, by way 15 of exarnples, Alzheimer's ~ ez~ce~ Spinal muscular Ataxia, mytonic dystrophy, Spinal Balbar Muscular Atrophy (SBMA), Kennedy syndrome, Parkinson's disease, Senile dementia, Circllln~ribed cerebral atrophy, Hlmtin~tQn's chorea, Cerebrocerebellar degeneration, Amaurotic family idiocy, Leukodystrophy, Familial myoclonus epilepsy, Hallervorden-Spatz ~li.eç~ç, Wilson's ~ice~ce, hepatolenticular degeneration, Westphal-20 Strumpell pseudosclerosis, Paralysis ~itsln~, Dystonia musculorum deformans, torsiondystonia, Hallervorden-Spatz ~i~ç~ce, Spasmodic torticollis, Cerebellar degenerations, Spinocerebellar degenerations, Friedrich's ataxia, Marie's hereditary ataxia, Amyotrophic lateral sclerosis, Progressive muscular atrophy, Progressive bulbar palsy, Primary lateral sclerosis, Werdnig-Hoffmarm disease, Wohlfart-Kugelberg-Welander25 syndrome, Hereditary spastic paraplegia, Progressive neural m~lcc~ r atrophy, Peroneal muscular atrophy (Charcot-Marie-Tooth) Hypertrophic hll~ ial neuropathy (Dejerine-Sottas), Leber's disease, retinitis pigmentosa and fragile X disorder.In another aspect of the present invention, t;x~fcs~ion cassettes or gene delivery constructs may be used to treat disorders char~cteri7e~1 by abnormal gene 30 expression, and inherited disorders caused by a known gene defect. In addition to a number of the disorders listed above, genes for defective enzymes or proteins have been identified, by way of example, for (1) Iysosomal storage disorders such as those involving ,B-hexosarninidase (Kornerluk etal., J. Biol. Chem. 261:8407-8413, 1986);
Myerowitz etal., Proc, Natl. Acad. Sci. flJSA) 82:5442-5445, 1985); Tsuji etal., N.
35 ~ngl. ~ Me~. 316:570-575, 1987), (2) for deficiencies in hypoxzlntlline phosphoribosyl transferase activity (the "Lesch-Nyhan" syndrome; Stout et al., Met. }inzymol. 151:519-=
WC~ 97J~3866 PCT/IJS96J16368 530, 1987), (3) for amyloid polyneuropathies (prealbumin; Sasaki et al., Biochem.
Biophys. Res. Commun. 125:636-642, 1984), (4) for ~17heimer's Disease (see, for example, Gont etal., Nature 349:704, 1991; Sherrington etal., Nature 375:754, 1995;
Levy-Labad etal., Science 269:973, 1995; Tanzi etal., Science 235:880-884, 1987;5 Goldgaber et al., Science 235:877-880, 1986), (5) for D-lrhenne~s muscular dystrophy (uncharacterized muscle protein; Monaco et al., Nature 323:646-650, 1987), and (6) for retinoblastoma (uncharacterized protein expressed in the retina and other tissues, Lee et al., Science 235:1394-1399, 1987; Friend et al., Nature 323:643-646, 1986).
Expression cassettes and gene delivery constructs may also be used to 10 study the "shiverer" mutation (myelin basic protein, Roach etal., Cell 42:149-155 (1987); Molineaux etal., Proc. Na~l. Acad. Sci. (USA) 83:7542-7546 (1986), and the "jumpy" mutation (proteolipoprotein, Nave et al., Proc. Natl. Acad. Sci. fUSA) 83:9264-9268 (1986); Hudson et al., Proc. Natl. Acad. Sci. fUS~l) 84:1454-1458 (1987)).
Expression cassettes and gene delivery constructs of the present 15 invention can also be used for trç~tment of acute injuries to the brain or peripheral nervous tissue, for exarnple from a stroke, brain injury, or spinal cord injury.Expression ç~c~ett~s and gene delivery constructs of the present invention may also be used in the ~r~ .cnt of disorders which require receptor modulation to increase or decrease tr~nsmit~çr uptake. Such disorders include 20 schizophrenia, obsessive-compulsive disorder, depression, and bipolar mood disorders.
As utilized within the context of the present invention, the term "trç~tment" refers to re~lucing or alleviating ~y~ OlllS in a subject, preventing symptoms from worsening or progressing, inhibition or elimin~tion of the causative agent, or prevention of the infection or disorder in a subject who is free th..~rl.~lll.
25 Thus, for example, tre~tment of infection includes destruction of the infecting agent, inhibition of or hllelr~l~ .lce with its growth or maturation, neutralization of its pathological 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 unb2l~nce~1 state disorder is "treated" by partially or wholly remedying the 30 imb~l~nce which causes the disorder or which makes it more severe.
The ~x~r~ssion c~ettes or gene delivery constructs of the present invention may be ~1mini~t~red by any one of several methods of ~-lmini~tration known in the art which account for the risk of degradation of the recombinant virus in the bloodstrearn and such that the virus retains its structure and is capable of infecting target 35 cells. Within one embodiment, ~lmini~tration may be accompli~hed by microinjection of the virus, alone or in a pharmzl~eutir~lly suitable carrier or diluent, through a CA 02234604 l998-04-l4 W O97tl3866 PCT~US96/16368 stereotactically-located pipette or syringe. Suitable locations vary with application, but include intraocular and brain injections.
Phar~m~ceutical carriers and diluents which are suitable for use within the present invention include, for example? water, lactose, starch, m~gne~iurn stearate, talc, 5 gum arabic, gelatine, polyalkylene glycols (e.g., polyethylene glycol), and the like. The ph~rmslreutical preparation 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 ph~rm~reutical p.c~aldLions can be subjected to 10 conventional ph~rm:~celltical adjuvants such as preserving agents, stabilizing agents, wetting agents, salts for varying the osmotic ~les~ t;, and the like. The present pharmaceutical ~ ualdlions may also contain other therapeutically valuable substances.
In another aspect of the present invention, ~r~ssion cassettes or the delivery constructs described herein may be delivered by chronic infusion using any 15 suitable method known in the art, including an osmotic minipump (Alza Corp.) or delivery through a time release or sll~tzlinecl release medium. Suitable time release or sl-ct~ine~l release systems include any methods 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 20 meçh~ni~m~ the composition may be injected into the cerebrospinal fluid via intrathecal or intraventricular injections, as well as into the brain substances and intraocular locations.
The expression cassette or gene delivery construct should be 7~lmini.ctcred in a therapeutically effective amount. A therapeutically effective amount 25 is that sufficient to treat the disorder. A therapeutically effective amount can be determined by in vitro ~ in~ent followed by in vivo studies. Expression of the inserted nucleic acid segment can be deterrnined in vitro using any one of the techniques described above. Expression of the inserted nucleic acid segment can be ~etermined in vivo using any one of several methods known in the art, including 30 immllnofluorescence using a fluoresceinated ligand.
In another aspect of the present invention, the t;~les~ion cassettes or gene delivery constructs described above may be incorporated into a phslrrnzlceutical composition. Preferably, the ph~ reutical composition contains one or more therapeutically effective doses of the cassette or construct in a suitable ph~rm:~relltical 35 carrier or diluent. Suitable pharmaceutical carriers and diluents are outlined above. A
theldpeuLically effective dose may be determined by in vitro ~p~ ent followed by W O 97/I3866 PCT~US96~16368 in vivo studies as described above. The composition may be ~tlmini~tered by any one Or the methods described above.
The following examples are provided by way of illustration, and not by way of limitation. Unless otherwise incli~t~-~, the specific protocols used in the 5 following examples are described in detail in Maniatis et al., supra, or Sambrook et al., supra.
E~AMPLE 1 GENERATION OF A RECOMBlNANT HERPESVIRUS VECTOR
1 0 WHIC~I EXPRESSES M 1 MUSCARINIC ACETYLCHOLINE RECEPTOR
A recombinant Herpesvirus vector which expresses the ml m-lc~rinic acetylcholine receptor (ml-AchR) was generated by homologous recombination between an HSV-l virus and a plasmid, pTKhml, which was constructed for this 15 purpose.
Briefly, pTKhml was prepared from the coding sequence for the hurnan ml-AchR gene and altered pTKSB. The coding sequence of ml-AchR was isolated as a 2.7 kb BamHI fragment from a plasmid cont~ining this sequence (Bonner et al., Science 237:527-532, 1987; see also generally Genbank Accession Nos.: M16404, 20 M16405, M16406, M16407, M16408, M16409) and inserted into a plasmid vector cont~ining a single BamHI cloning site. The coding sequence was re-isolated by digestion of that plasmid vector with EcoRI and HindIII.
pTKSB (Smiley et al., J. Vir. 61(8):2368-77 (1987)), which contains the HSV-I TK gene, was altered by insertion of a CMV promoter-co.l(~;..i..g fragment25 from the plasrnid pRc/CMV (Invitrogen Corporation). This fragment r~lt;s~llts the portion of the plasmid ~xtenfling from base 2û9 to base 1285 and co~ g the CMV
major imm~ te early promoter, a multicloning site, and a poly A addition site. The fragment was inserted into pTKSB by first digesting the plasmid with BamHI and then converting the BamHI site into a PacI site by the addition of adapter sequences. The 30 CMV promoter was oriented in the opposite direction to the TK promoter to reduce llallsc~ ional interference. The resulting plasmid (pTKSB cont~ining the CMV
promoter) was then digested with EcoRl and HindIII and ligated to the ml-AchR
coding se~uence which had also been digested with HindIII and EcoRl using collv~l,tion~l methods. This plasmid was referred to as pTKhml.
3~ pTKhml was then used to generate an HSV recombinant virus by in vivo homologous recombination. pTKhm l was cotransfected into Vero cells (ATCC
W O 97/13866 PCT~US96116368 Accession No. CRL 1587) along with an infectious HSV-1, vhsA. vhsA is a mutant HSV-l (FIG. la) ~see Smibert and Smiley, J: Vir. 64:3882, 1990) con1~inin~ the ,B-galactosidase gene in the UL4 1 gene coding sequence.
TK deficient recombinants were selected using bromodeoxycyt;dine.
S Following selection, virus isolates were plaque purified and tested for the CMV-ml-AchR insert by digestion with EcoRI, electrophoresis on a 1.1% agarose/TAE gel and hybridization to a radioactive probe. The probe was generated by incubating the mlAchr gene in buffer contzlining random hexamers of DNA to act as primers for extension 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 hybridization at 37~C in the presence of 50% formamide, 2X standard saline citrate, 5X Denhardt's solution, 1%
sodium dodecyl sulfate. Following incubation for 12 h, filters were washed extensively 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. lb), lacked a 2.1 kb EcoRI fragment Contzlinin~;
the endogenous TK gene and instead, contained a 4.6 kb EcoRI fr~mPnt which hybridized to the m 1 -AchR specific probe. Thus, it was det~rmin~cl that the neurotransmitter receptor gene was successfully introduced into the viral genome.
EXAMPI,E 2 DETECTION OF M 1 AcHR MRNA EXPRESSION FROM
RECOMBINANT VIRUSES
Expression of ml AchR transcripts from the CMV promoter was detected using a ribonuclease protection assay. (FIG. 2). Briefly, a labeled RNA probe was synthesized from 326 nucleotides (nt) from the T7 promoter of the plasmid BS/KS(-) (available from Stratagene Cloning Systems) comprising 265 nt of the 5' end of the mlAchr gene and ~6 nt of the 3' end of the CMV promoter. This probe targeted the 5' end of hurnan mlAchR mRNA as well as a portion of the CMV promoter. This labeledprobe was incubated with sarnples of total cellular RNAs isolated from Vero cells 2 to 18 hours post-infection (hpi) by vTKhml.
The reaction was then subjected to digestion by RNaseA and RNaseTl under conditions of high salt to inhibit digestion of double-strand RNA. Labeled probc that had hybridized to cellular RNA was subsequently identified following electrophoresis on an 8M urea/polyacrylarnide gel and visualized by autoradiography.
(FIG. 3). A protected RNA fragment of 265 nt which corresponded to transcription of W O 97/13866 PCT~US9G/16368 the insert from the CMV promoter was detected as early as 3 hours post-infection("hpi"), reached high levels by 8 hpi, and m~;nt~incd high levels until 18 hpi. (FIG. 3).
S ISOLATION OFICP4-RECOMBINANTS EXPRESSING THE MlAcHR GENE
Recombinants were generated by homologous recombination between two viruses: dl20, an ICP4(-) virus developed by DeLuca, (DeLuca et al., "Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Immediate Early li~egulatory Protein ICP4," J. Vir. 56:558-570, 1985), and vTE~hml (FIG. lb), prepared in Example 1. Briefly, the viruses were coinfected with E5 cells, an ICP4-expressing Vero cell line. The res~ in~ virus stock was selected for TK(-) mutants with bromodeoxycytidine, and clones were screened for their ability to grow on E5 cells, but not Vero cells.
Positive clones were then tested for the presence of the mlAchR gene by restriction digestion with EcoRI and Southern blot hybr;dization. One virus clone, referred to as vTKhm1-2 (FIG. lc), was found to both express mlAchR and form plaques only with E5 cells.
This recombinant was then used to generate a third recombinant, referred to as vTKhml-3 (FIG. ld), which is defective in both ICP4 and VHS ~:x~ession. E5cells were coinfected with vTKhm1-2 (FIG. lc) and vhsA, the HSV-l mutant that expresses ,B-ga}actosidase from its UL41 region. Bromodeoxycytidine was used to select against vhsA, and the r~sulting viral isolates were screened (a) for their ability to grow on ES cells, but not Vero cells, (b) for the expression of mlAchRs, and (c) for the e~ c;s~ion of ,B-galactosidase. These recombinants were referred to as vTKhm1-3 (FIG. ld).
DETECTION OF SURFACE RECEPTOR EXPRESSION FROM RECOMBINANT
VIRUSES IN VERO CELLS USING LIGAND BINDING ASSAYS
The e2~ ion of mlAchR from Vero cells infected with a multiplicity of infection of 10 with vTKhml-l, vTKhm1-2 and vTKhml-3 was compared using the ~3H]NMS ligand binding assay. Surface mlAchR were measured by incubating 35 infected Vero cells with 1 nM of the radiolabeled muscarinic receptor antagonist, n-methyl-scopolamine ([3H]NMS) at 37~C for 1 hour. After incubation with [3HlNMS, W O 97/13866 PCTrUS96/16368 the infected cells were washed three times with phosphate buffered saline, Iysed and counted in scintillation fiuid. Saturation curves were performed to determine the al,pl~xilllate number of mlAchRs represented by counts measured using lnM
[3H~NMS. (FIG. 4). Competitive inhibition by pi~ ,ille confirmed that these counts reflect specific binding of the ligand to mlAchRs.
Vero cells do not contain any endogenous mlAchRs, therefore any [3H]NMS binding above background represent receptors expressed from the recombinant virus. The expression of mlAchRs from each recombinant is shown.
(FIG. 4). The ICP4-mutant, vTKhrnl-2 infected Vero cells expressed 2-3 fold moremlAchRs 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 those infected with vTKhrn I -1 and at least 2-fold more than 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 t;x~l~,s~ion from vTKhm1-2 and vTKhml-3 are approximately the same. Receptor ex~les~ion from vTKhml plateaus by approximately 12 hpi, and by 36 hpi Vero cells infected with the replication competent vTKhm I -1 recombinant are dead.
DETECTION OF SUR~ACE RECEPTOR EXPRESSION FROM RECOMBINANT VIRUSES
The ~re~ion of mlAchR from E5 cells, ICP4(-~ Vero cells, infected with a multiplicity of infection of 10 with vTKhml-l, vTKhml-2 and vTKhrnl-3 wascompared using the same [3H]NMS ligand binding assay as in Example 4. (FIG. 5).
Complement~tion of the ICP4(-) mutation in vTKhrnl-2 and vTKhrnl-3 transfected E5 cells results in drastically reduced levels of mlAchRs. (FIG. 5). These results indicate that the increased explcs~ion levels in vTKhml-2 and vTKhml-3 infected Vero cells is related to lack of ICP4 ~x~!les~ion. The expression of ICP4 by the E5 cells allows the rccombinant viruses to replicate. (FIG. 11). This data filrther indicates that the lack of viral host-protein synthesis (VHS) exl,lc;ssion contributes to increased mlAchR
expression, since vTKhml-l and vTKhml-3 have higher expression levels than vTKhml-2 in E5 cells.
At 1 hpi and 12 hpi DNA was isolated from each of the infected Vero and E5 cell samples by standard methods and dotted onto nitrocellulose membrane in three fold dilutions. ~FIG. 11) vhsA infected Vero and E5 cell samples served as a control. These results demonstrate that vTKhm1-2 and vTKhml-3 samples replicatedin the E5 cell samples, but not in the Vero cell samples.
Southern blot analysis and immunofluorescence studies were performed to ensure that the recombinants, vTKhml-2 and vTKhml-3, were defective in ICP4 expression. Southern blot confilllled the presence of a 4.05 Icb deletion in ICP4. This deletion is characteristic of dl20, the ICP4(-~ HSV-l strain used to construct these recombinants. The e~ies:iion of the ICP4 product in Vero cells infected with the HSV-1 recombinants was assayed by indirect immunofluorescence using a monoclonal antibody directed against ICP4. Fluolescence micrographs 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 infected with vTKhml-l; vTKhml-2 and vTKhml-3 infected Vero cells did not express cletect~hle amounts of ICP4. (FIG. 9).
EXPRESSION OF MlACHRS FROM HSV-l RECOMBINANTS
IN PRIMARY CORTICAL NEURON CULTURES
Primary cortical neuron cultures, isolated from seven-day-old neonatal rats, were infected with either vTKhml-l, vTKhml-2, or vhsA at a multiple of infection of 3. At 12 hpi, the cultures were in-ub~t~l at 37~C with [3H]NMS for 1 hour. Inaddition, uninfected control cultures were assayed to measure the amount of endogenous mlAchR expressed in primary cortical neuron cultures. Atropine, an mlAchR antagonist which competes with [3H~NMS binding, was used to determine theamount of nonspecific ligand binding present in each sample. (FIG. 7).
In these assays, vTKhml-2 infected cells expressed 5 fold more mlAchRs than uninfected cultures, or approximately 38,000 surface receptors per cell as con~ d to 6,000 receptors on an uninfected cell. (FIG. 6). However, cells infecte~1 with vTKhml-l expressed less than a 2-fold increase in the amount of mlAchR
compared to uninfected cultures. (FIG. 6). vhsA infected cultures ~x~iessed fewer receptors than the uninfected cultures. (FIG. 6). Moreover, there were no cytopathic W O 97/13866 PCTrUS96/16368 effects evident in either vTKhm 1-2 infected neurons or the vTKhml -I infected neurons.
These results demonstrate that the recombinant viruses of the present invention reduce cytopathic effects associated with viral infection and provide heigh1e~t?d ~xl~es~ion of nucleic acid segment inserts.
A phosphatidylinositol turnover assay was performed on neuronal cells infected with each of vTKhml-1, vTKhml-2, and vTKhml -3. This assay demonstratesthat the mlAchR function to stim~ te second messenger systems. 10 d cultures of mouse cortical neurons were infected or mock-infected and then incubated prior to measurement of PI turnover using I uCi/ml [3H] inositol in inositol-free miniln~l 10 essential mcdium. Cultures were washed 3X in Hanks buffered saline solution. Cells were treated or mock-treated with 1 mM carbachol. After 45 rnin, the medium was removed, cells were washed once with HBSS, cold 3% perchloric acid was added, and inositol phosphate levels were determined exactly as described previously (Murphy et al., F~Sl~B J. 4:1624-1633, 1990). Second messengers were st;m~ tçcl S fold by 12 15 hpi in infected Vero cells. Second messengers were stimulated 4 fold in rat cortical neurons.
GENERATION OF A RECOMBINANT HERPESVIRUS VECTOR WHICH
EXPRESSES AN ANTISENSE S-~IT2A RECEPTOR SEQUENCE
A. Materials and Conskuction Methods 1. Vector Construction Plasmid pTKSR2(-) (which is based on pUC 19) consists o~ a Cytomegalovirus (CMV) immediate early promoter, an entire coding region of rat 5-HT2 lecel)lol gene in an ~nti~n~e orientation (see Julius et al., PN~S 87:928-932, 1990; see also Genbank Accession No. M30105), and a polyA sequence from the bovine growth hormone gene. The promoter-~nti~çn~e S-HT2 receptor gene se~uence 30 and poly A region is framed by a Pvu II fragment of the HSV-1 tk gene.
A HSV-1 kos mutant skain vhs A was utilized for construction of recombinant virus. Briefly, in vhs A, a genome region responsible for shutting off host-cell macromolecules synthesis has been deleted. The virus was propagated in African Green Monkey kidney cells (Vero, ATCC Accession No. C~L 1587) cultured with 35 MEM mçdillm with 10% fetal bovine serum (Gibco BRL) at 37~C and 10% CO2.
W O 97113866 PCTrUS96/16368 The recombinant virus was constructed using homologous recombination described above. Briefly, viral DNA from vhs A and pTKSR2(-) plasmid DN~ were cotransferred into Vero cells ntilizing calcium phosphate precipitation. One hundred micrograms of broma-D-oxycytidine (Sigma) was added to the culture to select for tk(-) S virus. Generated virus was collected and diluted with medium to infect Vero cells in 96-well dishes. After 48 hours, medium con~inin~ released virus and cellular debris from the 96 wells were blotted on a membrane followed by a hybridization with a 32p labelled probe made from the 5-HT2 receptor gene. Virus from positive wells werethen plaque purified three times and insertion of the antisense construct was confirmed 10 by Southern blotting and DNA polymerase chain reaction.
2. Preparation of Neuronal Cultures Rat primary cortical cultures were prepared from postnatal day 1 Long-Evans rats. Briefly, each rat was sacrificed by decapitation under halothane anesthesia.
The cerebral cortices were ~ ect~d and minced with iridectomy scissors into fine15 fragments in a brain dissecting buffer solution cont71inin~; Ca2+/Mg2+ free Hank's B~l~nc~e-l Salt Solution (HBSS, Gibco). The fr~gm~ntc were transferred into 3 ml of a solution with 0.16% trypsin (Sigrna) and 0.03% DNase I (Boehrirlger Mannheim) at37~C for 10 minlltes The tissue was then gently dissociated with a 5 ml plastic pipette followed by addition of 2 ml ice cold fetal bovine serum to stop the trypsin activity.
20 The cell suspension was centrifuged and the cell pellets were resuspended in MEM
supplement with 4 mM L-g~ut~min~, 16 mM NaHCO3, 20 mM HEPES and 10% FBS.
After two washes with fresh medium, 2 x 106 cells in 2 ml medium were plated onto poly-L-lysine pretreated glass coverslips in 35 mrn dishes. The culture was incubated in a 5% CO2 incubator at 37~C. The medium was replaced on the following day with fresh 25 medium col.~;~i..;..~ 20 mM cytosine arabinofuranoside (Ara-C, ~igma) to inhibit the proliferation of nonneuronal cells.
3. R~ase Protection Assav Two RNA probes were generated by in vitro transcription. Briefly, probe A was 600 nt with a sequence identical to 5' end sense strain of rat 5-HT2a 30 receptor gene. Probe B was 620 nt in length and spans a region including 300 nt sequence of CMV promoter followed by the first 320 nt sequence of 3' end rat 5-HT2a receptor gene. An RNase protection kit was used and the assay was carried out following the m~nl-f~c*lrer's instruction. The digested hybridized probes were run on 5% Acrylamide gel and exposed on film overnight.
CA 02234604 l998-04-l4 4. Li~and Bindins~Assavs Cells from primary cortical cultures were collected and were freezed-thawed three times before being used for the ligand assays. 106 cells were incubated with 1 nM[3H]ketanserin hydrochloride (85.1 C~i/mmol) or 5nM[3H~mesulergine 5 (Amersham, 750 Ci/mmol) (both from NEN, Du Pont) or 4.5 nMI3H]N-methyl-scopolamine (NMS, 75 Ci/mmol) in Tris buffer (170 mM, pH 7.5) for one hour at room temperature. The non-specific bindings in these assays were less than 20% determined by adding 2,000-fold concentrations of non-isotope-labelled sarne ligands into the correspondin~; incubation solutions. Following the incuh~tion, the cells were washed 10 3 x 5 min in the same buffer at 4~C and re~usl~ellded in 1 ml of ~ormula 963 (NEN) for counting of radioactivity in a Beckman LS 2800 liquid scintillation counter.
B. Results The recombinant virus, vTKSR2(-), co~ ir~g an antisense construct for 15 the rat 5-HT gene was used to infect Vero cells at multiplicity of infection (moi)=3.
Total RNA from the infected cells was collected at 7, 17 and 24 hours postinfection.
Probe A gave rise to a protected band which e~uals to the full length of the probe while probe B was only partially protected and gave rise to a band of 320 nt, the size of transcribed portion. The amount of ~nSi~n~e transcripts increased with the time of 20 postinfection up to 17 hours and declined thereafter.
To prove that the transcribed antisense mRNA could block the ssion of endogenous 5-HT2a receptors in neurons, primary cultures of rat cerebral cortex were used for ligand binding assays following the viral infections. At 7 hours postinfection of vTKSR2(-)(moi=3), the neuronal cultures were double labelled with a 25 monoclonal antibody against microtubule-associated protein type 2 (MAP-2), a neuron-specific marker, and a polyclonal antibody against HSV-l virus. More than 90% ofcells in the cultures were labelled by MAP-2 antibody indicating their neuronal nature.
Among the MAP-2 positive cells, more than 80% of them were also showed immunoreactivity of HSV-1 antigens, indicating that a large population of the neurons 30 were infected by the recombinant virus. Following the infection with vTKSR2(-) or the parental virus vhs A at moi=3, neurons were incubated with [3H3ketanserin or [3H]NMS
at either 7 hours, 17 hours and 48 hours postinfection. The radioactivity of bound [3H]ketanserin was significantly reduced in cultures infected with vTKSR2(-) comp~rin~ to the VHS A infected ones. The reduction of binding in neuronal cultures 35 peaked at 17 hours and declined in 48 hours postin~ection. In contrast, there was no difference in binding of [3H]NMS between the vTKSR2(-) and VHS A infected W O 97/13866 PCTfUS96/16368 cultures. Furthermore, using ~3H3mesulergine, a ligand more specific for 5-HT2c receptors, to label the infected cultures, a minor reduction of binding was observed with a marginal significance of statistics.
IN VIVO ADMIN~STRATlON
Long-Evans rats were utilized as subjects to determine the degree of infectivity of recombinant Herpesvirus vectors. Briefly, the rats were ~n~ctheti~cl with an intraperitoneal injection of sodium pentobarbitol (Somnotol, 40 mg/kg), and placed into a stereotaxic appal~Lus. A craniotomy was performed over the appropriate brain structure, and the Herpesvirus vector SHT2 described above in Example 8 was injected.
In the case of one rat, a total of 10 ul at a titer o~ 108 pfu/ml was injected into the hippocampus over a period of 20 mimlt~ The rat was then sutured and treated with a topical antibiotic (chloramphenicol, 1%). After 4 days, the rat was sacrificed and the hippocampal region sectioned and stained with an immunohistochemical to recognize the viral envelope. The results are shown in Figure 12B. Briefly, infection can be seen in a substantial number of cells within the needle track.
Another rat was injected in the cortex with 200 ul at a titer of 5 x 107 pfulml over a period of 90 m;n~ltes As above, the rat was then sutured and treated with a topical antibiotic. After 2 days the rat was sacrificed and the cortex sectioned and stained. Results are shown in Figure 12A. Briefly, strong labelling (and thus, infection of cells) can be seen throughout the periphery of the cortex.
CONSTRUCTION AND ASSAY OF NEURON-SPECIFIC VECTORS
A. M~tçri~l~ and Construction Methods All restriction endonucleases, media, fetal bovine serum (FBS), nerve growth factor (NGF) and fine chemicals were obtained from GIBCO BRL Life Technologies Inc. (Burlington, Ontario~. Unless otherwise noted, cloning procedures followed standard procedures (Sambrook etal., "Molecular Cloning: A laboratory m~nu~l," 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
Plasmid pON134 contains the HSV-l (strain KOS~ latency-associated transcript promoter (LAPl) (Selden etal., Mol. Cell. Biol. 6:3173-3179, 1986; Ho and W O 97tl3866 PCT~US96tl6368 Mocarski, Proc. Nal'l Acad. Sci US~ 86:7596-7600, 1989). Briefly, the LAPl promoter is located immediately u~ t;;anl of the large primary approximately 8.5kb LAT RNA transcript and about 650 bp U~iL~ of the 5' ends of the small LAT RNAs.
As shown in Figure 13, this promoter COllt~ S a typical TATA box and several 5 characteristic eukaryotic transcription regulatory elements, such a cyclic AMP-response elements, a CATT box, and two Spl sites. One region of the LAP promoter (betweenabout -161 and about -259~ is particularly preferred for conferring a neuronal cell-type specific activity.
The LAP1 promoter was cloned from pON134 by digestion of the 10 plasmid with PvuI and a 609 bp DNA fragment containing the LAP l promoter from ~2 to -608 (Batchelor and O'Hare, ~ Yirol. 66:3573-3582, 1992; McGeoch et al., J. Gen.
ViroL 69:1531-1574, 1988) was inserted at the Hinc II site of the pBluescript KS (+) vector (Stratagene, LaJolla, Califorr~ia). The minimum LAPl promoter used in ~}is study was constructed by a filrther SstII deletion of the upstrearn sequences at -259.
Dimer neuronal restrictive silencer elements were constructed by ~nne~lin~ two synthetic oligonucleotides essentially as described by Mori et al. (Neuron 9:45-54, 1992), S36~: CAAAGCCATT TCAGCACCAC GGAGAGTGCC TCTGC
(Sequence 1.I~. No. 2) and S36B: GCAGAGGCAC TCTCCGTGGT GCTGAAATGG
CTTTG (Sequence I.D. No. 3), followed by ligating the double-stranded DNA
2û fragments and inserting them into a pBluescript KS(+) vector at the Hinc II site. The correct sequences and orientations of dimer silencers were examined and verif1ed by standard double-stranded dideoxynucleotide sequencing.
The human cytomegalovirus (hCMV) major immediate early gene enhancer from -113 to -601 (Boshart etal., Cell 41:521-530, 1985) was cloned from 25 pRc/CMV (lnvitrogen, San Diego, California) by digestion of the hCMV major immediate early promoter with Ban I and Hinc II so that the TATA box of the promoter was deleted. The 488 bp enhancer fragment was inserted at the Hinc II site of pBluescript KS(+).
A 21kb BamHI-EcoRI human growth hormone (hGH) gene fragment 30 including its own polyadenylation signal was cloned out from a plasmid pOGH (Selden et al., Mol. Cell. Biol. 6:3173-3179, 1986).
B. Cells. Transfections and hGH Assay Vero and rat pheochromocytoma (PC12) cells were obtained from the 35 ATCC. Briefly, Vero cells are monkey kidney cells which are capable of supporting HSV-I propagation in vitro. PC12 cells are neural crest derived rat W O 97/13866 PCTrUS96116368 phaeochromocytoma cell line. In response to nerve growth factor, PC12 cells stopdividing and differentiate into neuron-like cells with many properties characteristic of peripheral nervous tissue.
Vero cells were propagated in Oulbecco's Modified Eagle Medium 5 (DMEM) with 10% FBS and 10% inactive horse serum. Difrele,lliation of PC12 cells induced by NGF at a concentration of 150 ng/ml was performed at 16 hours post-transfection. Transfection of Vero and PC12 cells with the plasmid constructs on six-well dishes with 3 x 105 cells per well was performed lltili7inl7 the calcium phosphate-me~ t.ocl precipitation method essentially as described by Sambrook et al., supra. The 10 amount of DNA used in the transfection was titrated to 5 ug per well. The linear response range of hGH accllm~ cl in the medium was det~rm;ned hetween 48 to 96 hours after transfection for both cell types and therefore, the media were collected at 48, 72 and 96 hours after transfection. The amount of hGH secreted into the medium was measured with a solid-phase two-site radioimmunoassay Icit under the conditions 15 recommen-le~l by the m~nllf~c$ure (Nichols Tn~tit~lt~ Diagnostics, Los Angeles, California). Data were collected from at least two sets of transfection experiments and each transfection was performed in triplicates.
C. Chimeric ~ ei,~ion cassettes with LAPl promoters and NRSE silencer 20 elements To selectively alter LAP 1 promoter activity only in neuronal cells, several synthetic NRSE dimers were placed upstream of the LAPl promoter in all possible orientations (Figure 14A) and tested in both Vero and PC12 cells by transient expression assays. As shown in Figure 14~3, the NRSE monomer (pSA/LAP/HGH) 25 su~lc;ssed the LAPl promoter activity by only 20% in Vero cells and had no effect on the promoter activity in PC12 cells as co~ al~d to the LAPl promoter without NRSE
(pLAP/HGH). Moreover, when the chimeric LAPl promoters with the head-to-tail NRSE dimers in either orientation (pS2A/LAP/HGH and pS2B/LAP/HGH) were introduced into Vero cells, the promoter activity decreased to 60 to 80% relative to the 30 parental plasmid construct (pLAP/HGH). These NRSE dimers however, did not inhibit LAPl promoter activity in PC12 cells. In addition, chimeric LAP1 promoters with either head-to-head or tail-to-tail NRSE dimers (pS2H/LAP/HGH and pS2T/LAP/HGH) showed somewhat lower promoter activity (about 60%) as compared to the construct without NRSE and had no cell-type p.~felence for Vero or PC12 cells.
35 From these results it was concluded that an NRSE element efficiently suppressed the HSV-1 LAP1 promoter in non-neuronal cells and an NRSE dimer was more effective than a monomer when placed in a head-to-tail orientation. Therefore, the chimeric promoter construct pS2B/LAP/HGH was chosen for further experiments.
The hCMV enhsm~çr element from -113 to -601 of the hCMV major IE
promoter was placed either u~ c~ll of the LAP 1 promoter (pE/LAP/HGH) or at the 3' S end of the hGH reporter gene (pLAP/HGH/E). The resulting chimeric promoter constructs were introduced into both Vero and PC12 cells. As shown in Figure 15B, the LAP1 promoter activity was stimulated by the enhancer in both cell types as compared to the basal level activity of the basic LAP I promoter construct pLAP/HGH in each cell line, which indicates that the enh~n~ ~r element worked in both cell types. Moreover, in 10 Vero cells, the LAP I promoter activity was up-regulated by about 2.5 fold regardless of the position of the enhancer. By contrast, in PC12 cells, when the hCMV enhz~nl~er was placed at the 3' end of the hGH gene (pLAP/HG~/E), the enh~n-er activity was five times higher than when the enhancer was upstrearn of the LAP l promoter (pE/LAP/HGH).
To evaluate the potential cell-type l~ler~r~llce contributed by different promoters, the hCMV major immediate early (IE) promoter with its own enh~nt~er was included as a control. As shown in Figure l5B, in Vero cells the hCMV major IE
promoter itself had a much higher activity (fourteen fold) than the LAP1 promoter, whereas in PC12 cells it had the same activity as the LAP1 promoter. These results suggest that the relative hCMV enhancer activity in dirr~lclll cell types was well dependent on the promoter to which it was linked. In addition, since the enh~n~relement itself showed no promoter activity in either Vero or PC12 cells, the chimeric LAP1 promoter must have been enh~nce~ as a result of interactions between the LAP1 promoter and the hCMV enh~ncer element. These results demon~tr~ted that the HSV-1 LAP1 promoter could be up-regulated in both neuronal and non-neuronal cells by an enhancer from the hCMV major IE promoter. However, the level of enhancement of the LAPI promoter in neuronal cells was dependent on the position of the enh:lnr~r element relative to the promoter.
In summ~ry, the HSV-1 LAP1 promoter can be regulated by other gene regulatory sequences either to ~7U~lC3s the promoter activity in non-neuronal cells, or to enhance its activity in both neuronal and non-neuronal cells.
D. Chimeric e~ es~,ion cassettes with LAP I promoters. NRSE silencer elements and hCMV enhancers The chimeric LAP1 promoter construct cont~inin~ the silencer dimer, pS2B/LAP/HGH, was used as a backbone for further studies. Briefly, this construct W O 97~13866 PCT~US96/16368 contained a head-to-tail NRSE dimer upstream of the LAP1 promoter and had the lowest promoter activity among the constructs tested in Vero cells (Fig. 14B). The hCMV enhancer element was added either ~ l of the LAP 1 promoter (pS2B/E/LAP/HGH), or at the 3' end of the hGH reporter gene (pS2B/LAP/HGH/E), as~ 5 shown in Fig. 16A. When pS2B/E/LAP/HGH and pS2B/LAP/HGH/E constructs were introduced into Vero and PC12 cells, they all showed a higher level of promoter activity in both cell types than the construct pS2B/LAP/~GH, which lacks the enh~ncer element. Moreover, in Vero cells, the NRSE dimer no longer ~ essed the LAP1 promoter activity in the presence of the enhancer (lF ig. 1 6B). Thus, the hCMV enh~nc.er 10 activity was dominant over the silencing activity of the NRS~ dimer element when they were combined. Interestingly, the enhancer also showed position effects on the LAPl promoter activity in the presence of the NR~E dimer. When it was placed at the 3' end of the hGH reporter gene (pS2B/LAP/HGH/E), it showed a stronger ~nh~nfçment of the LAPl promoter in PC12 cells than in Vero cells as demonstrated previously (~ig. lSB).
15 By contrast, when it was placed at the 5' end of the LAP 1 promoter (pS2B/E/LAP/H~H), it showed an unusually high level of activity in Vero cells, whereas in PC12 cells it showed only slight enhancing activity.
~. The Chimeric LAPI Promoter Activity in the Differentiated PC12 Cells 20 Tn~ cecl bYNGF
Nerve growth factor (NGF) is important for the m~ te.~sln~e of neuronal cell survival. In the presence of NGF, PC12 cells undergo various physiological changes and differentiate into neuron-like cells with many characteristics of peripheral nervous tissue.
In order to test the responses of LAPl promoters in NGF in~ e-1 PC12 cells, chimeric LAPl promoter constructs were first transfected into PC12 cells and then cell ~liLrt;~ iation was incln~ed by treatment with NGF. As shown in Fig. 17B, the LAP1 promoter activity increased about four fold in response to NGF, whereas the chimeric LAPl promoter with the hCMV enh~n~er element (pLAP/HGH/E) showed 30 two fold increased activity. Interestingly, the hCMV major IE promoter also increased its activity to the sarne extent as the LAPl promoter in response to NGF tre~tment in ~ PC12 cells, which suggests that the up-regulation of the LAP1 promoter in the differenti~ted PC12 cells was part of an overall stim~ tion of transcription in response ~ to NGF.
From the foregoing, it will be evident that although specific embo~imenf~ of the invention have been described herein for the purposes of W O 97/13866 PCT~US96/16368 illustration, various modifications may be made without deviating from the spirit and scope of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Cynader, Max Tufaro, Franci s (ii) TITLE OF INVENTION: METHOD OF USING, PROCESS OF PREPARING.
AND COMPOSITION COMPRISING RECOMBINANT HERPESVIRUS VECTORS
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(B) FILING DATE: 11-OCT-199 (C) CLASSIFICATION:
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Claims (52)
1. An expression cassette capable of expressing a sequence of interest, comprising one or more neuronal specific silencer elements, a promoter element operably linked to a sequence of interest, and an enhancer, wherein said enhancer and silencer elements are positioned such that they are not adjacent to one another.
2. The expression cassette according to claim 1 wherein said silencer element is a neuronal restrictive silencer element.
3. The expression cassette according to claim 1 wherein said promoter element is selected from the group consisting of CMV, SV40, herpes promoters andadenovirus promoters.
4. An expression cassette, comprising a LAT promoter element operably linked to a sequence of interest, followed by an enhancer.
5. The expression cassette according to claim 4, further comprising a neuronal-specific silencer element, wherein said enhancer and silencer elements are positioned on said vector such that they are not adjacent to one another.
6. The expression cassette according to claim 5 wherein said neuronal-specific silencer element is a neuronal-restrictive silencer element.
7. The expression cassette according to claim 1 or 4 wherein said sequence of interest is selected from the group consisting of antisense sequences, genes which encode disease-associated antigens, genes which encode immunologically active molecules, requirement genes, and genes which encode toxic proteins.
8. A gene delivery construct which contains an expression cassette according to any one of claims 1-7.
9. The gene delivery construct according to claim 9 wherein said construct is a Herpesvirus vector.
10. A host cell which contains an expression cassette according to any one of claims 1-7.
11. A method for producing a protein, comprising:
(a) introducing an expression cassette according to any one of claims 1-7 which directs the expression of a protein of interest, or a gene delivery construct according to claim 8, into a host cell; and (b) culturing said host cell under conditions, and for a time sufficient, to permit expression of said protein.
(a) introducing an expression cassette according to any one of claims 1-7 which directs the expression of a protein of interest, or a gene delivery construct according to claim 8, into a host cell; and (b) culturing said host cell under conditions, and for a time sufficient, to permit expression of said protein.
12. The method of claim 11, further comprising the step of purifying said protein.
13. A method for introducing a selected sequence of interest into neuronal cells of a warm-blooded animal, comprising administering to said animal an expression cassette according to any one of claims 1-7, or a gene delivery construct according to claim 8.
14. A method for introducing a selected sequence of interest into an in vitro culture containing neuronal cells, comprising introducing an expression cassette according to any one of claims 1-7, or a gene delivery construct according to claim 8, into an in vitro culture containing neuronal cells.
15. A recombinant Herpesvirus vector capable of directing the expression of a G protein linked receptor gene.
16. A recombinant Herpesvirus vector capable of directing the expression of an antisense transcript of a G protein linked receptor gene.
17. The recombinant Herpesvirus vector according to claim 15 or 16 wherein said recombinant virus is deficient for thymidine kinase expression.
18. The recombinant Herpesvirus vector of claim 17 wherein the gene encoding the G protein linked receptor or the antisense transcript is inserted in a TK locus of said recombinant virus.
19. The recombinant Herpesvirus vector of claim 15 wherein the G-protein linked receptor gene is selected from the group consisting of a gene which encodes an adrenergic receptor and a human M1 muscarinic acetylcholine receptor gene.
20. The recombinant Herpesvirus vector according to claim 16 wherein the antisense transcript is that of a 5-HT2 receptor gene.
21. The recombinant Herpesvirus vector according to claim 15 or 16 wherein said virus is deficient in expression of a virion host shut-off protein (VHS).
22. The recombinant Herpesvirus vector according to claim 15 or 16 wherein said vector contains one or more neural specific silencer elements, a promoter operably linked to a sequence of interest, and an enhancer, wherein said enhancer and silencer are positioned such that they are not adjacent to one another.
23. The recombinant Herpesvirus vector according to claim 15 or 16 wherein said virus is replication defective.
24. The recombinant Herpesvirus vector of claim 23 wherein said virus is deficient in expression of ICP4 protein.
25. A recombinant Herpesvirus vector which is deficient in the expression of the virion host shut off protein VHS and thymidine kinase.
26. A recombinant Herpesvirus vector which is deficient in the expression of ICP4 and thymidine kinase.
27. A recombinant Herpesvirus vector which is deficient in the expression of the virion host shut off protein VHS, ICP4 and thymidine kinase.
28. A method of treating mammals for neurological disorders, comprising administering to a mammal a composition comprising a recombinant Herpesvirus vector, according to any one of claims 15-27, in combination with a pharmaceutically acceptable carrier or diluent.
29. The method of claim 28 wherein said composition is administered by stereotactical microinjection.
30. The method of claim 28 wherein said composition is administered by a time release mechanism, a sustained release mechanism, or chronic infusion.
31. Ex vivo mammalian cells infected with a recombinant Herpesvirus vector according to any one of claims 15-27.
32. A pharmaceutical composition comprising a recombinant virus according to any one of claims 15-27 and a pharmaceutically acceptable carrier or diluent.
33. A process of producing recombinant Herpesvirus vector virus with low cytopathicity, comprising:
culturing mammalian cells with a first recombinant Herpesvirus vector containing a G protein linked receptor gene and a second recombinant Herpesvirus vector 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.
culturing mammalian cells with a first recombinant Herpesvirus vector containing a G protein linked receptor gene and a second recombinant Herpesvirus vector 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.
34. The process of claim 33 wherein said G protein linked receptor gene is inserted in the thymidine kinase locus.
35. The process of claim 34 wherein the G protein linked receptor gene is an adrenergic receptor gene.
36. The process of claim 34 wherein the G protein linked receptor gene is a human M1 muscarinic acetylcholine receptor gene.
37. The process of claim 33 wherein the first recombinant virus is deficient in the expression of thymidine kinase.
38. The process of claim 33 wherein the first recombinant is deficient for expression of the virion host shut-off protein VHS.
39. The process of claim 33 wherein the first recombinant virus is vhsA.
40. The process of claim 33 wherein the second recombinant virus is d120.
41. The process of claim 33 wherein the protein required for replication is ICP4.
42. The process of claim 33 wherein the recombinant virus is deficient in the expression of the virion host shut off protein VHS and thymidine kinase.
43. The process of claim 33 wherein the recombinant virus is deficient in the expression of ICP4 and thymidine kinase.
44. The process of claim 33 wherein the recombinant virus is deficient in the expression of the virion host shut off protein VHS, ICP4 and thymidine kinase.
45. A recombinant Herpesvirus vector with an in vitro cytopathicity generally less than about 3%.
46. The recombinant virus of claim 44 wherein the cytopathicity is in about the range of 0.1 % to 1.0%.
47. The recombinant virus of claim 45 wherein the cytopathicity is in about the range of 0.001% to 0.1%.
48. A recombinant Herpesvirus vector capable of directing the expression of a G protein linked receptor, said Herpesvirus vector being capable of expressing on the surface of an infected cell greater than 10,000 receptors/cell.
49. The virus of claim 48 capable of expressing on the surface of an infected cell about the range of 25,000-200,000 receptors/cell.
50. The virus of claim48 capable of expressing on the surface of an infected cell about the range of 200,000 to 400,000 receptors/cell.
51. The virus of claim 48 capable of expressing on the surface of an infected cell greater than about 400,000 receptors/cell.
52. A recombinant Herpesvirus vector according to any one of claims 15-27, for use in the manufacture of a medicament for treating mammals with neurological disorders.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/540,692 US5763217A (en) | 1993-11-10 | 1995-10-11 | Method of using, process of preparing and composition comprising recombinant herpesvirus vectors |
| US08/540,692 | 1995-10-11 | ||
| PCT/US1996/016368 WO1997013866A2 (en) | 1995-10-11 | 1996-10-11 | Recombinant herpes virus vectors for expression in neuronal cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2234604A1 true CA2234604A1 (en) | 1997-04-17 |
Family
ID=29405973
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2234604 Abandoned CA2234604A1 (en) | 1995-10-11 | 1996-10-11 | Recombinant herpes virus vectors for expression in neuronal cells |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2234604A1 (en) |
-
1996
- 1996-10-11 CA CA 2234604 patent/CA2234604A1/en not_active Abandoned
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