Replication competent, avirulent Herpes simplex virus as a vector for neural and ocular gene therapy

Degenerative diseases of the retina are a leading cause of vision loss in the United States, affecting approximately two million people each year. The replacement of a defective gene by gene therapy provides one approach for treating individuals having ocular neuronal degeneration where the defective gene has been identified. Several factors, however, suggest that the replacement of a specific gene in a patient might not be effective. For example, many of the conditions are autosomal dominant, and placing a normal copy of the gene into the cells would not correct the defect. As an alternative, replication competent, avirulent, ribonuclease reductase deficient Herpes simplex virus can provide the means to deliver therapeutic polypeptides in a continuous manner to affected cells. Such therapeutic polypeptides include growth factors, neurotrophins and cytokines.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to methods for using recombinant Herpes 
simplex virus to treat neuronal degeneration. More particularly, this 
invention relates to methods for treating neuronal degeneration, including 
ocular neuronal degeneration, by administering a Herpes simplex virus that 
expresses a therapeutic gene in infected cells, but does not express 
ribonucleotide reductase. 
2. Related Art 
Degenerative diseases of the retina are a leading cause of vision loss in 
the United States, affecting approximately two million people each year. 
VISION RESEARCH--A NATIONAL PLAN, 1994-1988, Report of the National 
Advisory Eye Council, National Institutes of Health, National Eye 
Institute, pages 41 and 55; Berson, Proc. Nat'l Acad. Sci. USA 93:4526 
(1996). Certain retinal degenerations, such as retinitis pigmentosa are 
clearly inherited and can be classified as autosomal dominant, autosomal 
recessive, or X-linked. Humphries et al., Science 256:804 (1992). 
Another major cause of retinal degeneration is macular degeneration. 
Macular degeneration primarily affects people older than 65 and is a 
leading cause of blindness in this group. The majority of macular 
degeneration has not yet been linked to genetic factors and the cause of 
macular degeneration remains unknown for most patients. VISION RESEARCH--A 
NATIONAL PLAN, 1994-1988, Report of the National Advisory Eye Council, 
National Institutes of Health, National Eye Institute, pages 41 and 55. 
Recent studies on retinitis pigmentosa have shown that mutations in several 
genes coding for proteins in the phototransduction pathway are involved. 
Most are in rhodopsin but the peripherin/rds (retinal degeneration slow) 
gene, cyclic GMP-phosphodiesterase gene, and the RCC1 guanine nucleotide 
exchange factor may also be involved. See, for example, Dryja et al., 
Proc. Nat'l Acad. Sci. USA. 88:6481 (1991); Inglehearn et al., Hum. Mol. 
Gen. 1:41-45 (1992); Al-Maghtheh et al., Hum. Mol. Gen. 3:205 (1994); 
Meindl et al., Nature Genetics 13:35 (1996). In humans, retinitis 
pigmentosa has been mapped to other genetic loci indicating that several 
other genes may be involved in the disease. Humphries et al., Science 
256:804 (1992). Recent results have also identified a mutation in the 
human peripherin/rds gene in the cause of autosomal dominant macular 
dystrophy in three families. McLaughlin et al., Nature Genetics 4:130 
(1993); Gal et al., Nature Genetics 7:64 (1995). These results suggest 
that both retinitis pigmentosa and macular degeneration may have a common 
underlying mechanism, defects in phototransduction proteins, and that 
common strategies for treatment might be possible. Mutations in the rod 
C-GMP phosphodiesterase .beta.-2 subunit gene have been implicated in 
autosomal dominant stationary night blindness, again suggesting that 
defects in phototransduction cascade proteins cause several retinal 
degenerative diseases. Tsang et al., Science 272:1026 (1996). 
Gene therapy is certainly one strategy that might be used for treatment in 
individuals where the defective gene has been identified. Several factors, 
however, suggest that the replacement of a specific gene in a patient 
might not be effective. A clinical test to determine the mutation would be 
required. This is not yet widely available and would likely require that 
specialized diagnostic centers be established. Moreover, a method for the 
delivery of the gene to the photoreceptor cells is needed. Finally, many 
of the conditions are autosomal dominant, and placing a normal copy of the 
gene into the cells would not correct the defect. Treatment would have to 
"turn off" the mutant allele, which would be very difficult to do in all 
cells. 
Recent studies of the effects of neurotrophins (i.e., growth factors for 
nerves) have provided information that may be used to develop a gene 
therapy treatment for several retinal degenerations which would not 
require the identification of the mutation. For example, injection of bFGF 
or brain derived neurotrophic factor (BDNF) into the vitreous delays 
retinal degeneration and light-induced retinal degeneration in albino 
rats. LaVail et al., Proc. Nat'l Acad. Sci. USA 89:11249 (1992); 
Faktorovich et al., Nature 347:83 (1990). The effect, however, is 
transient and continued administration of the factor is required for long 
term preservation. If genes for these factors could be delivered to the 
eye, continued synthesis should be therapeutically useful. 
Therefore, a need exists for a means to deliver therapeutic polypeptides or 
proteins on a continuous basis to treat neuronal degeneration. 
SUMMARY OF THE INVENTION 
In one aspect, the present invention provides methods for treating neuronal 
degeneration, including ocular neuronal degeneration, by administration of 
a recombinant Herpes simplex virus that stimulates expression of a 
therapeutic gene in infected cells. 
The present invention also provides a replication competent Herpes simplex 
virus that expresses in infected cells a therapeutic gene and a functional 
product of the .sub..gamma. 34.5 gene, but does not express functional 
ribonucleotide reductase. 
DETAILED DESCRIPTION 
1. Definitions 
In the description that follows, a number of terms are utilized 
extensively. Definitions are herein provided to facilitate understanding 
of the invention. 
Structural gene. A DNA sequence that is transcribed into messenger RNA 
(mRNA) which is then translated into a sequence of amino acids 
characteristic of a specific polypeptide (protein). 
Promoter. A DNA sequence which directs the transcription of a structural 
gene to produce mRNA. Typically, a promoter is located in the 5' region of 
a gene, proximal to the start codon of a structural gene. If a promoter is 
an inducible promoter, then the rate of transcription increases in 
response to an inducing agent. In contrast, the rate of transcription is 
not regulated by an inducing agent if the promoter is a constitutive 
promoter. 
Enhancer. A promoter element. An enhancer can increase the efficiency with 
which a particular gene is transcribed into MRNA irrespective of the 
distance or orientation of the enhancer relative to the start site of 
transcription. 
Complementary DNA (cDNA). Complementary DNA is a single-stranded DNA 
molecule that is formed from an mRNA template by the enzyme reverse 
transcriptase. Typically, a primer complementary to portions of MRNA is 
employed for the initiation of reverse transcription. Those skilled in the 
art also use the term "cDNA" to refer to a double-stranded DNA molecule 
derived from a single mRNA molecule. 
Expression. Expression is the process by which a polypeptide is produced 
from a structural gene. The process involves transcription of the gene 
into mRNA and the translation of such mRNA into polypeptide(s). 
Cloning vector. A DNA molecule, such as a plasmid, cosmid, phagemid, or 
bacteriophage, which has the capability of replicating autonomously in a 
host cell and which is used to transform cells for gene manipulation. 
Cloning vectors typically contain one or a small number of restriction 
endonuclease recognition sites at which foreign DNA sequences may be 
inserted in a determinable fashion without loss of an essential biological 
function of the vector, as well as a marker gene which is suitable for use 
in the identification and selection of cells transformed with the cloning 
vector. Marker genes typically include genes that provide tetracycline 
resistance or ampicillin resistance. 
Expression vector. A DNA molecule comprising a cloned structural gene 
encoding a foreign protein which provides the expression of the foreign 
protein in a recombinant host. Typically, the expression of the cloned 
gene is placed under the control of (i.e., operably linked to) certain 
regulatory sequences such as promoter and enhancer sequences. Promoter 
sequences may be either constitutive or inducible. 
Recombinant host. A recombinant host may be any prokaryotic or eukaryotic 
cell which contains either a cloning vector or expression vector. This 
term is also meant to include those prokaryotic or eukaryotic cells that 
have been genetically engineered to contain the cloned gene(s) in the 
chromosome or genome of the host cell. For examples of suitable hosts, see 
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) 
["Sambrook"]. 
RR.sup.- or RR deficient HSV. A RR deficient, or "RR.sup.-," HSV is a 
Herpes simplex virus that does not produce functional ribonucleotide 
reductase. 
Therapeutic gene. In the present context, a "therapeutic gene" is a gene 
that encodes a product used to treat a neuronal degenerative disease or 
disorder. Suitable therapeutic genes include growth factors, neurotrophins 
and cytokines. 
2. Overview 
Replication deficient viral vectors are frequently suggested for use in 
gene therapy because of safety concerns associated with using replication 
competent viruses. The problem with replication deficient viruses is that 
they infect one cell, and cannot propagate through a tissue or a larger 
area. Thus, if delivery is not efficient, only a limited number of cells 
are transformed. This is a serious limitation, particularly in the area of 
neural and ocular delivery, because replication is required for a virus to 
cross a synapse. 
Replication deficient Herpes simplex viruses have been used as gene therapy 
vectors with the amplicon system. This system requires an amplicon (which 
contains the origin of replication and a packaging sequence from the 
virus, as well as the gene of interest), a helper cell line, and a helper 
cell virus. The cell line contains a gene which is essential for viral 
replication, so that when the helper cell line is infected with the helper 
virus, replication occurs. Some of the virus particles produced from this 
infection have packaged the amplicon, and some have packaged the helper 
cell virus, so it is then necessary to separate one from the other. A 
recent variation of this method uses a set of overlapping clones from the 
helper virus which has a deletion in the packaging sequence. This system 
results in packaging of only the amplicon. Previous attempts to deliver a 
gene to various parts of the eye have used adenoviruses, adeno-associated 
viruses, and Herpes simplex virus. So far, researchers have only been able 
to "label" retinal cells via subretinal injection, which causes retinal 
detachment in the area of the injection. 
The mutant hrR3 vector has a deletion in the ribonucleotide reductase (RR) 
gene. The RR deficient virus replicates well in tissue culture, and to 
some extent in animals, but does not cause any significant pathology in 
animals. The inventors utilized a vector with the lacZ gene inserted into 
the area of the RR deletion, and used intravitreal injection, anterior 
chamber injection, and corneal scarification to introduce the vector into 
the eyes of mice and rats. After allowing sufficient time for gene 
expression, the animals were sacrificed, and examined for gene expression 
in the eye as evidenced by blue color. Corneal scarification resulted in 
gene expression in the area of scarification. When the vector was injected 
into the anterior chamber, the iris turned blue. Intravitreal injection 
resulted in columns of blue cells from the inner and outer nuclear layers 
to the ganglia. These columns provide evidence that the virus can cross 
synapses. 
In addition, the inventors have inserted the gene for basic fibroblast 
growth factor (bFGF) into these vectors in the area of the ribonucleotide 
reductase deletion. Studies show that cells infected with the recombinant 
HSV vector express bFGF. This successful gene insertion was accomplished 
after three years of effort on the part of the inventors. 
Initial attempts to create a viral vector incorporating a fibroblast growth 
factor gene in the area of the RR deletion were unsuccessful due to the 
natural limitations of creating such a vector. Such limitations include 
the fact that there must not be an overlapping gene present in the 
insertion that is essential. Furthermore, deletion of both the RR gene and 
a second "non-essential" gene may render the virus replication incompetent 
or so restricted in its growth that it cannot survive. 
Initial constructs by the inventors attempted to insert the basic 
fibroblast growth factor (bFGF) gene into the latency-associated 
transcript (LAT) gene, which is expressed only in neurons. A HSV fragment 
encoding LAT and several kilobases of flanking DNA was cloned. The LAT 
gene was then cut just downstream of the LAT promoter, and the bFGF cDNA 
inserted to produce a plasmid, with the intention that the bFGF gene would 
then be controlled by the neuron specific LAT promoter. This plasmid was 
then transfected with purified hrR3 vector and attempts were made to 
plaque purify the LAT-bFGF recombinant. No simple screens to identify the 
recombinant existed, so plaques were chosen and their DNA analyzed by 
Southern blotting. Although a total of 500 plaques were screened, all 
results were negative. Although Southern blotting of the entire 
transfection mixture showed that bFGF gene had inserted into the hrR3 
vector, no pure viral vectors containing the bFGF gene could be isolated. 
It was postulated that either the insertion of the bFGF gene disrupted 
some essential function of the virus, or more likely, that insertion of 
the bFGF gene resulted in a second disruption of the virus, that combined 
with the deletion in the RR gene, crippled viral growth to such an extent 
that a pure virus containing the bFGF gene insertion could not be 
isolated. Similar attempts utilizing a CMV-bFGF plasmid, as described in 
Example 2 infra, which was then inserted into the nonessential 
glycoprotein C gene and transfected with purified hrR3 DNA were 
unsuccessful. It was postulated that the combination of a glycoprotein C 
gene and RR gene deletion crippled viral growth in the manner previously 
described. 
3. Construction of Replication Competent Herpes Simplex Virus Vectors That 
Express a Foreign Gene 
Various methods for gene therapy are available. These include placement of 
transfected cells carrying the gene into the host, delivery of naked DNA 
to muscle cells, use of cationic lipid carriers, and use of viruses such 
as retroviruses, adenoviruses, and Herpes simplex virus (HSV). See, for 
example, Meindl et al., Nature Genetics 13:35 (1996); Mulligan, Science 
260:926 (1993); Rosenberg et al., Science 242:1575 (1988); LaSalle et al., 
Science 259:988 (1993); Wolff et al., Science 247:1465 (1990); Breakfield 
and Deluca, The New Biologist 3:203 (1991). HSV seems particularly suited 
for delivery of genes to neurons since infection of neurons is a normal 
part of the virus life cycle. Additional advantages of HSV-based vectors 
include the ability to deliver genes to non-dividing cells, and the 
ability to infect many cell types in both animals and humans. Fields 
(ed.), VIROLOGY, pages 527-561 (Raven Press 1985). 
The inventors have identified genes in Herpes simplex virus-l (HSV) that 
are involved in virulence. Brandt et al., J. Gen. Virol. 72:2043 (1991); 
Herold et al., J. Gen. Virol. 75:1211-1222 (1994); Visalli and Brandt, 
Virology 185:419 (1991). In particular, the HSV type 1 ribonucleotide 
reductase (RR) gene was found to be required for corneal virulence. Brandt 
et al., J. Gen. Virol. 72:2043 (1991). The RR gene was also found to be 
required for acute retinal disease. Brandt et al., Arch. Virol. 142:883 
(1997). 
In addition, Goldstein and Weller, J. Virol. 166:41 (1988), have shown that 
RR deficient mutants are severely compromised in the ability to replicate 
at 39.5.degree. C. in vitro. Such mutants, therefore, are less likely to 
propagate in an infected host who has a fever. Furthermore, RR deficient 
HSV is hypersensitive to acyclovir and ganciclovir, and consequently, 
RR.sup.- HSV is responsive to antiviral therapy. Thus, RR deficient HSV 
have attenuated neurovirulence and are susceptible to antiviral therapy in 
the event that the host has viral encephalitis. 
Martuza et al., U.S. Pat. No. 5,585,096 (1996), have described the 
production of replication competent HSV to kill tumor cells. Although the 
HSV vector is RR deficient, the vector also has a mutation in the 
.sub..gamma. 34.5 gene. As a result, the vector produces neither RR nor 
the product of the .sub..gamma. 34.5 gene. While the present invention 
contemplates an HSV vector having such a double mutation, the methods 
described herein do not require the use of such a vector. Suitable HSV 
vectors of the present invention are incapable of expressing the RR gene. 
Useful RR.sup.- vectors include those that express a functional product 
of the .sub..gamma. 34.5 gene, as well as vectors that are incapable of 
expressing a functional .sub..gamma. 34.5 gene product. 
Those of skill in the art are capable of constructing HSV vectors that are 
RR deficient. See, for example, Goldstein and Weller, J. Virol. 62:196 
(1988). HSV-1 DNA can be obtained, for example, from commercial sources 
such as the American Type Culture Collection (ATCC No. VR-260). HSV-2 DNA 
can be obtained, for example, from commercial sources such as The American 
Type Culture Collection (ATCC No. VR540). 
A method for constructing a RR.sup.- vector that expresses a foreign gene 
is provided in Example 2 herein. In the example, a vector was produced 
that expresses a bovine bFGF gene under the control of the Human 
Cytomegalovirus Immediate Early gene promoter. Additional genes that are 
suitable for expressing in cell lines or for gene therapy include, but are 
not limited to, genes encoding: acidic fibroblast growth factor (aFGF; 
FGF-1); glial cell line-derived neurotrophic factor; brain-derived 
neurotrophic factor; ciliary neurotrophic factor; nerve growth factor; 
interleukin-1.beta.; superoxide dismutase; extracellular matrix proteins 
(collagens, fibronectins, integrins); cell adhesion molecules; 
neurotransmitter receptors; ornithine amino transferase; prostaglandin 
synthesis regulation proteins; trabecular meshwork proteins; hypoxanthine 
phosphoribosyltransferase; tyrosine hydroxylase, and prostaglandin 
receptors. 
Nucleotide sequences encoding these polypeptides are known to those of 
skill in the art. For example, Abraham et al., Science 233:545 (1986), 
disclose the nucleotide sequence of bovine bFGF, while the nucleotide 
sequence of human bFGF is disclosed by Abraham et al., EMBO J. 5:2523 
(1986). Mergia et al., Biochem. Biophys. Res. Commun. 164:1121 (1989), 
provide the nucleotide sequence of the human aFGF gene. The nucleotide 
sequence of the rat glial cell line-derived neurotrophic factor is 
described by Springer et al., Exp. Neurol. 131:47 (1995). Maisonpeirre et 
al., Genomics 10:558 (1991), provide the nucleotide sequences of human and 
rat brain-derived neurotrophic factor, while Arab et al., Gene 185:95 
(1997), disclose the amino acid sequence of bovine brain-derived 
neurotrophic factor. Rat ciliary neurotrophic factor is described by 
Stocki et al., Nature 342:920 (1989). The nucleotide sequence of the human 
ciliary neurotrophic factor gene is disclosed by Negro et al., Eur. J. 
Biochem. 201:289 (1991), Lin et al., Science 246:1023 (1989), and by Lam 
et al., Gene 102:271 (1991). Ulrich et al., Nature 303:821 (1983), provide 
a comparison of human and murine coding regions of beta-nerve growth 
factor genes. The nucleotide sequence of bovine interleukin-.beta.1 is 
disclosed by Leong et al., Nucl. Acids Res. 16:9054 (1988), while Bensi et 
al., Gene 52:95 (1987), provide the nucleotide sequence of the human 
interleukin-1.beta. gene. 
DNA molecules encoding such polypeptides can be obtained by screening cDNA 
or genomic libraries with polynucleotide probes having nucleotide 
sequences based upon known genes. Standard methods are well-known to those 
of skill in the art. See, for example, Ausubel et al. (eds.), SHORT 
PROTOCOLS IN MOLECULAR BIOLOGY, 3rd Edition, pages 2-1 to 2-13 and 5-1 to 
5-6 (John Wiley & Sons, Inc. 1995). 
Alternatively, DNA molecules encoding growth factors can be obtained by 
synthesizing DNA molecules using mutually priming long oligonucleotides. 
See, for example, Ausubel et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR 
BIOLOGY, pages 8.2.8 to 8.2.13 (1990). Also, see Wosnick et al., Gene 
60:115 (1987); and Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR 
BIOLOGY, 3rd Edition, pages 8-8 to 8-9 (John Wiley & Sons, Inc. 1995). 
Established techniques using the polymerase chain reaction provide the 
ability to synthesize DNA molecules at least two kilobases in length. 
Adang et al., Plant Molec. Biol. 21:1131 (1993); Bambot et al., PCR 
Methods and Applications 2:266 (1993); Dillon et al., "Use of the 
Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," 
in METHODS IN MOLECULAR BIOLOGY, Vol. 15: PCR PROTOCOLS: CURRENT METHODS 
AND APPLICATIONS, White (ed.), pages 263-268, (Humana Press, Inc. 1993); 
Holowachuk et al., PCR Methods Appl. 4:299 (1995). 
DNA molecules encoding growth factors can also be obtained from commercial 
sources. For example, a clone of the human aFGF gene can be obtained from 
the American Type Culture Collection (ATCC No. 53335). 
High titer stocks of recombinant HSV can be obtained from infected 
mammalian cells using standard methods. For example, recombinant HSV can 
be prepared in Vero cells, as described by Brandt et al., J. Gen. Virol. 
72:2043 (1991), Herold et al., J. Gen. Virol. 75:1211-1222 (1994), Visalli 
and Brandt, Virology 185:419 (1991), Grau et al., Invest. Ophthalmol. Vis. 
Sci. 30:2474 (1989), and by Brandt et al., J. Virol. Meth. 36:209 (1992). 
Also see, Brown and MacLean (eds.), HERPES SIMPLEX VIRUS PROTOCOLS (Humana 
Press 1997). 
4. Use of RR Deficient HSV to Produce Proteins in Cell Lines 
To express the foreign polypeptide, the DNA sequence encoding the 
polypeptide must be operably linked to regulatory sequences that control 
transcriptional expression in an expression vector and then, introduced 
into a host cell. In addition to transcriptional regulatory sequences, 
such as promoters and enhancers, expression vectors can include 
translational regulatory sequences and a marker gene which is suitable for 
selection of cells that carry the expression vector. 
Expression vectors that are suitable for production of a foreign protein in 
eukaryotic cells typically contain (1) prokaryotic DNA elements coding for 
a bacterial replication origin and an antibiotic resistance marker to 
provide for the growth and selection of the expression vector in a 
bacterial host; (2) eukaryotic DNA elements that control initiation of 
transcription, such as a promoter; and (3) DNA elements that control the 
processing of transcripts, such as a transcription 
termination/polyadenylation sequence. 
Foreign proteins of the present invention are preferably expressed in 
mammalian cells. Examples of mammalian host cells include African green 
monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells 
(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK21; ATCC CRL 
8544), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary 
cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH.sub.1 ; ATCC CCL82), 
HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548) 
SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murine 
embryonic cells (NIH-3T3; ATCC CRL 1658). 
For a mammalian host, the transcriptional and translational regulatory 
signals may be derived from viral sources, such as adenovirus, bovine 
papilloma virus, simian virus, or the like, in which the regulatory 
signals are associated with a particular gene which has a high level of 
expression. Suitable transcriptional and translational regulatory 
sequences also can be obtained from mammalian genes, such as actin, 
collagen, myosin, and metallothionein genes. 
Transcriptional regulatory sequences include a promoter region sufficient 
to direct the initiation of RNA synthesis. Suitable eukaryotic promoters 
include the promoter of the mouse metallothionein I gene [Hamer et al., J. 
Molec. Appl. Genet. 1:273 (1982)], the TK promoter of Herpes virus 
[McKnight, Cell 31:355 (1982)], the SV40 early promoter [Benoist et al., 
Nature 290:304 (1981)], the Rous sarcoma virus promoter [Gorman et al., 
Proc. Nat'l Acad. Sci. USA 79:6777 (1982), the cytomegalovirus promoter 
[Foecking et al., Gene 45:101 (1980)], and the mouse mammary tumor virus 
promoter. See, generally, Etcheverry, "Expression of Engineered Proteins 
in Mammalian Cell Culture," in PROTEIN ENGINEERING: PRINCIPLES AND 
PRACTICE, Cleland et al. (eds.), pages 163-181 (John Wiley & Sons, Inc. 
1996). 
Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA 
polymerase promoter, can be used to control fusion gene expression if the 
prokaryotic promoter is regulated by a eukaryotic promoter. Zhou et al., 
Mol. Cell. Biol. 10:4529 (1990); Kaufman et al., Nucl. Acids Res. 19:4485 
(1991). 
An expression vector can be introduced into host cells using a variety of 
techniques including calcium phosphate transfection, liposome-mediated 
transfection, electroporation, and the like. Preferably, transfected cells 
are selected and propagated wherein the expression vector is stably 
integrated in the host cell genome to produce stable transformants. 
Techniques for introducing vectors into eukaryotic cells and techniques 
for selecting stable transformants using a dominant selectable marker are 
described, for example, by Ausubel and by Murray (ed.), GENE TRANSFER AND 
EXPRESSION PROTOCOLS (Humana Press 1991). 
General methods for expressing and recovering foreign protein produced by a 
mammalian cell system is provided by, for example, Etcheverry, "Expression 
of Engineered Proteins in Mammalian Cell Culture," in PROTEIN ENGINEERING: 
PRINCIPLES AND PRACTICE, (Cleland et al., eds.), pages 163-181 
(Wiley-Liss, Inc. 1996). 
5. Use of RR Deficient HSV to Treat Ocular and Neural Diseases 
As briefly discussed above, various polypeptides are useful for treatment 
of ocular and neural diseases. For example, subretinal or intravitreal 
injection of a number of growth factors, cytokines and neurotrophins 
(bFGF, brain derived growth factor, interleukin-1.beta.) have been shown 
to restore specific functions to retinal or retinal pigment epithelial 
cells and to retard photoreceptor cell death in various animal models of 
retinal degeneration. Faktorovich et al., Nature 347:83 (1990); LaVail et 
al., Proc. Nat'l Acad. Sci. USA 89:11249 (1992). Moreover, Faktorovich et 
al., Nature 347:83 (1990), have shown that the rate of photoreceptor 
degeneration can be significantly slowed by an intraocular injection of 
bFGF in Royal College of Surgeons rats that have inherited retinal 
dystrophy. Intraocular administration of bFGF also protects photoreceptors 
from light-induced degeneration in albino rats, a noninherited form of 
retinal degeneration. LaVail et al., Ann. N.Y. Acad. Sci. 638:341 (1991). 
Similarly, Unoki and LaVail, Invest. Ophthalmol. Vis. Sci. 35:907 (1994), 
showed that intravitreal injection of brain-derived neurotrophic factor, 
ciliary neurotrophic factor and basic FGF at least transiently protects 
rat retina from ischemic injury. Also see, Zhang et al., Invest. 
Ophthalmol. Vis. Sci. 35:3163 (1994). Furthermore, implants with either 
basic FGF or acidic FGF can rescue adult retinal ganglion cells from 
axotomy-induced cell death in rats. Sievers et al., Neurosci. Lett. 76:157 
(1987). Also see, GROWTH FACTORS AS DRUGS FOR NEUROLOGICAL AND SENSORY 
DISORDERS--SYMPOSIUM NO. 196, CIBA Foundation Symposia Series (1996). 
Accordingly, recombinant HSV vectors that express growth factors, cytokines 
and neurotrophins are suitable for treating ocular neuronal degenerative 
diseases and disorders, including retinitis pigmentosa, macular 
degeneration, glaucoma, optic neuropathies, and trauma. The recombinant 
vectors described herein are also suitable for treating diseases and 
disorders involving neuronal degeneration. Such diseases include 
Alzheimer's Disease, stroke, trauma, and retinal degeneration. 
The efficacy of any particular HSV vector that expresses a therapeutic 
protein can be tested in an appropriate animal model of a neuronal 
degenerative disease or disorder. As an illustration, see Example 5. 
In general, it is desirable to administer the highest dose possible without 
inducing toxicity. The actual dose will vary depending on the volume of 
vector preparation that can be introduced and this varies depending on the 
site of administration. The titer of stocks can be as high as 
1.times.10.sup.9 -1.times.10.sup.10 Plaque Forming Units (PFU)/ml. In the 
human eye it should be possible to give at least 100 .mu.l per injection, 
which is equivalent to 1.times.10.sup.8 1.times.10.sup.9 PFU per 
injection. In the brain, this volume should also be possible. Peripheral 
or intravenous injection could deliver a much higher dose. Multiple 
administration may also make it possible to increase the dose. 
Administration of recombinant HSV to a patient can be via injection, 
including intravenous and intravitreal injection, and by infusion into the 
cerebrospinal fluid, or by other means well known in the art. 
A composition comprising recombinant Herpes simplex viruses of the present 
invention can be formulated according to known methods to prepare 
pharmaceutically useful compositions, whereby viruses are combined in a 
mixture with a pharmaceutically acceptable carrier. A composition is said 
to be a "pharmaceutically acceptable carrier" if its administration can be 
tolerated by a recipient patient. Sterile phosphate-buffered saline is one 
example of a pharmaceutically acceptable carrier. Other suitable carriers 
are well-known to those in the art. See, for example, REMINGTON'S 
PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and GILMAN'S 
THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing 
Co. 1985). 
For purposes of therapy, a recombinant Herpes simplex virus and a 
pharmaceutically acceptable carrier are administered to a subject in a 
therapeutically effective amount. A combination of virus and a 
pharmaceutically acceptable carrier is said to be administered in a 
"therapeutically effective amount" if the amount administered is 
physiologically significant. An agent is physiologically significant if 
its presence results in a detectable change in the physiology of a 
recipient subject. In the present context, an agent is physiologically 
significant if its presence inhibits the progress of neuronal 
degeneration. 
The present invention, thus generally described, will be understood more 
readily by reference to the following examples, which are provided by way 
of illustration and are not intended to be limiting of the present 
invention.

EXAMPLE 1 
Distribution of the RR.sup.- Vector Virus (hrR3) in Rat Tissues 
To determine the cell types that are infected with an HSV vector, rats were 
exposed to hrR3, an RR deficient mutant virus. Construction of the hrR3 
vector is described by Goldstein and Weller, J. Virol. 62:196 (1988). The 
hrR3 virus has the E. coli lacZ gene inserted into the RR locus. The 
expression of the lacZ gene results in the formation of blue colored 
plaques in the presence of X-gal substrate. 
In these studies, rats were injected with hrR3 virus, and seven days later, 
tissues were collected for analysis. The tissues injected included the 
visual cortex, vitreous, anterior chamber, and cornea. Analysis of six 
treated rat brains revealed X-gal staining at the site of the injections 
(i.e., primary visual cortex) and in the lateral geniculate nucleus (LGN). 
This result shows that a foreign gene can be delivered and expressed using 
this vector, and that the vector can spread to neurons quite distant from 
the infection site. Uptake was specific since lacZ staining was only seen 
in the visual cortex and LGN. Other sites had blue cells, but this was due 
to endogenous lacZ expression as determined from control brains of rats 
that had not received the virus. As determined by morphology, up to about 
300 neurons were labeled in each positive LGN. When 1.times.10.sup.6 
-1.times.10.sup.7 PFU were injected intraviterally, lacZ staining was seen 
in the retinal pigment epithelium, non-pigmented ciliary body epithelium, 
photoreceptor cells, and cells in the inner nuclear layer. Cells in the 
optic nerve were also labeled on occasion. Injection of 1.times.10.sup.6 
-1.times.10.sup.7 PFU into the anterior chamber resulted in labeling in 
the iris and the angle of the eye (trabecular meshwork cells). Uninjected 
eyes or PBS injected eyes are negative for lacZ staining. After corneal 
scarification, only cells along the corneal scratches are labeled. 
EXAMPLE 2 
Delivery of BFGF to Sites of Neuronal Damage Using Transfected Cells 
A study was performed to test the possibility that the efficacy of basic 
fibroblast growth factor (bFGF) treatment could be improved by prolonging 
the exposure of axotomized lateral geniculate nucleus (LGN) neurons to 
bFGF. Briefly, quail teratocarcinoma cells (QT6) were obtained from 
American Type Culture Collection (ATCC, Baltimore, Md.), and grown in M199 
media (Gibco BRL; Bethesda, Md.) containing 10% tryptose phosphate broth, 
2.5 units/ml penicillin, 2.5 .mu.g/ml streptomycin (Gibco BRL), 1% DMSO 
and 5% fetal calf serum (Hyclone, Logan, Utah). To produce the 
bFGF-positive (QBF) cells, a 616 base pair BamHI and EcoRV fragment 
containing the wild type CDNA for the bovine bFGF gene was cleaved from 
the plasmid pBSbFGF and inserted into the multiple cloning region of the 
plasmid pCDNA3 (Invitrogen, CA). The nucleotide sequence of the bFGF gene 
is disclosed by Abrahams et al., Science 233:545 (1986). 
The expression of the bovine bFGF gene was driven by the human 
cytomegalovirus immediate early enhancer-promoter (CMV). This plasmid also 
included the neomycin resistance gene, allowing for selection in 
eukaryotic cells using G418. QT6 cells were transfected with pCDNA3-bFGF 
using a low CO.sub.2, low pH, modified calcium phosphate precipitation 
protocol. See, for example, Chen and Okayama, Mol. Cell. Biol. 7:2745 
(1987). Two days later, transfected QBF cells and control QT6 cells were 
replated at identical densities and placed in medium containing 600 
.mu.g/ml G418. Cells were passaged every three days until all the control 
cells were dead (approximately three weeks). Stably transfected QBF cells 
were then maintained in medium containing 300 .mu.g/ml G418. 
Stable incorporation of the bovine bFGF gene into the transfected quail 
cells was demonstrated by Southern Blot analyses using a 616 base pair 
EcoRI fragment which included the entire bovine bFGF cDNA as a probe. To 
determine whether the CMV-bFGF construct was transcribed in the QBF cells, 
total cellular RNA from QBF and QT6 cells was analyzed by Northern 
blotting analysis, using the bFGF cDNA as a probe. A transcript of the 
predicted size (.about.800 base pairs) was readily detected in the RNA 
from QBF cells, but not in RNA from the QT6 cells. The ability of the QBF 
cells to make bFGF protein was determined by Western blot analysis. When 
probed with an anti-bovine bFGF antibody, three bands of 18 KD, .about.22 
KD and .about.16 KD were seen in the QBF cell lane, compared to a single 
18 KD band seen in this range in the QT6 cell lane. Based on the amount of 
protein present in the control lanes, it was estimated that the QBF cells 
made approximately 0.14-1.4 fg of bFGF protein per cell. Moreover, the 
expression of the bovine bFGF gene appeared to give a mitogenic advantage 
to the transfected cells, suggesting that functional bFGF protein was 
being made by the QBF cells. 
Rats having a lesion of the visual cortex received implants of quail cells 
transfected with the bFGF gene, and were compared with rats that received 
either a single administration of bFGF protein directly, 0.9% saline, or 
untransfected quail cells. Rats were sacrificed at one, two, or four weeks 
after the cortical lesion was made, and the total number of surviving 
lateral geniculate nucleus neurons and their mean cross-sectional areas 
were determined. 
The results of these experiments showed that, at one week postoperatively, 
the numbers of surviving LGN neurons in QBF cell implanted rats were 136% 
greater than in saline-treated control rats. However, at two and four 
weeks, neuronal numbers in QBF cell implanted rats were similar to 
saline-treated controls. 
In contrast to the transient neuroprotective effects on neuronal cell 
numbers, the effects on mean neuronal cross-sectional areas were 
long-term, lasting up to four weeks. These surviving neurons exhibited 
mean areas that were consistently larger than the controls (19%, 48% and 
35% larger at one, two and four weeks). Surprisingly, the neuroprotective 
effects of the QBF cell implants were different from those seen after a 
single administration of the recombinant human bFGF protein alone, which 
prevented neuronal loss for at least three months, but did not reduce cell 
atrophy. Accordingly, quail teratocarcinoma cells transfected with the 
bovine bFGF gene and implanted at the site of a visual cortex lesion are 
capable of reducing both neuronal death and atrophy of axotomized LGN 
neurons. 
EXAMPLE 3 
Insertion of the bFGF Gene Into the Ribonucleotide Reductase Locus 
As discussed supra, prior attempts to insert the bFGF gene into the LAT and 
gC locus of HSV DNA failed. The reasons are not clear, but may be due to 
the deletion of two genes in the virus (RR and LAT, or RR and gC). 
An attempt was then made to insert the bFGF gene into the RR locus to 
produce a virus with only a single gene disruption. To accomplish this, 
the CMV-bFGF gene was transferred to a plasmid, pMAK. The pMAK plasmid 
contains the HSV-1 RR gene interrupted by the E. coli lacZ gene. The 
CMV-bFGF gene was inserted into the middle of the lacZ coding region. The 
resulting plasmid, "PMAK bFGF" thus carried the CMV-bFGF gene flanked by 
lacZ sequences which are then flanked by HSV-1 RR sequences. 
The CMV-bFGF gene was inserted into the lacZ gene of the hrR3 virus. 
Recombinants were identified by selecting for colorless plaques. The 
pMAK-bFGF sequences were inserted into hrR3 by co-transfecting purified 
hrR3 DNA with pMAK bFGF into African green monkey kidney (Vero) cells. The 
resulting virus pool was then plaqued on Vero cell monolayers in the 
presence of X-gal and infected cells were screened for colorless plaques. 
A total of 14 such plaques were picked and replaqued two more times. Thus, 
each of the 14 plaques were purified three times and the stock viral 
preparations were completely free of blue plaque virus (wild type hrR3). 
In order to identify viruses carrying the bFGF gene, viral DNA was 
purified, digested with EcoRI or EcoRV, and analyzed using Southern blot 
analysis with bFGF probe. Since EcoRI releases the bFGF sequence, a 600 
base pair EcoRI fragment should be identified on the blots. EcoRV cleaves 
once in PMAK bFGF so this digest should contain a bFGF containing fragment 
that extends into the flanking viral DNA confirming the gene was 
integrated into the hrR3 genome. Of the 14 plaques, two gave the predicted 
fragment patterns. These two viruses were designated HSV-2526 and 
HSV-5042. The presence of the bFGF gene in the viruses was confirmed by 
re-isolating DNA and repeating the Southern analysis. Moreover, analysis 
of RNA from cells infected with either HSV-2526 or HSV-5042 demonstrated 
the production of bFGF RNA transcripts. In addition, immunoblot analysis 
showed that HSV-2526- and HSV-5042-infected cells produce bFGF protein. 
EXAMPLE 4 
Localization of BFGF Gene Expression in Animals Infected With RR Deficient 
HSV 
To identify the distribution of bFGF expression, rats are injected with the 
bFGF-virus and at 1, 3, 5, and 7 days post-injection, the eyes are 
collected and processed for histology and immunohistochemical localization 
of bFGF antigens. Brandt et al., Curr. Eye Res. 13:755 (1994). For 
histology, eyes are fixed in phosphate buffered saline containing 10% 
formalin for three hours. The tissues are then embedded in paraffin, 
sectioned, and stained with hematoxylin/eosin by standard methods. Brandt 
et al., Curr. Eye Res. 13:755 (1994). For immunohistochemistry, eyes are 
embedded in OCT compound, snap frozen and sectioned with a cryomicrotome. 
They are then stained for bFGF using commercially available anti-bovine 
bFGF antisera (Dako, Carpinteria, Calif.) followed by alkaline phosphatase 
conjugated secondary antibody (Sigma, St. Louis, Mo.). Appropriate 
controls, such as non-specific antisera and secondary antibody only, are 
included, as are uninfected eyes. 
The measurement of bFGF expression presents a problem in distinguishing 
between synthesis from the endogenous host gene and synthesis from the 
CMV-bFGF gene. Although increased bFGF protein may be detected levels in 
infected cells by immunohistochemistry as described above, available 
antibodies do cross react with rodent and bovine bFGF. 
The use of reverse transcription-polymerase chain reaction (RT-PCR) 
provides reliable detection of CMV-bFGF gene expression. To make the assay 
specific for the delivered gene, 5' primers are used that anneal to 
sequences in the CMV promoter between the TATAA box and the bFGF coding 
region. The 3' primer anneals in the coding region of the bovine bFGF 
gene. .beta.-Actin is amplified as a control. This method corrects for the 
differences in the size of the tissue sample used for analysis, as 
described previously. Brandt et al., Curr. Eye Res. 13:755 (1994). 
Retina and ciliary body/iris are examined for bFGF RNA. Tissues are sampled 
at various times (days 3 or 4, 1 week, 2 weeks, 1 month, and 2 months). If 
the expression of the CMV-bFGF gene decreases to low levels before 1-2 
months, the bFGF virus is re-administered. Expression and the effect on 
retinal degeneration are monitored. A total of 3-5 rats are analyzed at 
each time point. 
EXAMPLE 5 
Analysis of HSV Expressing Brain-Derived Neurotropic Factor in Animal Model 
Systems 
Vectors expressing brain-derived neurotrophic factor (BDNF) can be tested 
in in vivo models of retinal degeneration, which are available in both 
rats and mice. An advantage of the light-induced degeneration in the 
albino rat model is that one can control the onset of degeneration. LaVail 
et al., Proc. Nat'l Acad. Sci. USA 89:11249 (1992). In this way, it is 
possible to be precise about when treatment and onset of disease begin. 
Rats are obtained from a commercial source, such as Harlan Sprague Dawley 
(Indianapolis, Ind.), and are maintained on standard food and water ad 
libitum as well as on a 12 hr:12 hr (light-dark) cycle at less than 2.0 
footcandle illuminance. Retinal degeneration is induced by increasing the 
light levels to 115 to 200 footcandles, and maintaining continuous light 
exposure, as described previously. LaVail et al., Proc. Nat'l Acad. Sci. 
USA 89:11249 (1992). 
For treatment, rats are anesthetized with intramuscular ketamine (84 mg/kg) 
and intraperitoneal xylazine (12 mg/kg). The viruses are delivered by 
intravitreal injection by inserting a 32 ga. needle in the right eye 
through the sclera into the vitreous just posterior to the corneal scleral 
junction on the nasotemporal side. A 5 .mu.l suspension of virus 
(1.0.times.10.sup.7 to 1.0.times.10.sup.8 pfu/ml) is injected using a 
Hamilton syringe. The needle is left in the eye for 60 seconds to 
equilibrate pressure, and then removed. The rats are monitored until 
recovery and then returned to animal care. 
In initial studies, rats are sacrificed at 7 and 14 days for evaluation. If 
these early points suggest a positive effect, studies can be extended to 
months to evaluate long term effects. Typically, there will be three 
groups: hrR3 control, media control, and the HSV-2526 or HSV-5042 virus. 
An additional control is provided by the contralateral (left) eye, because 
an RR mutant virus does not cross into the contralateral eye. Additional 
rats are sacrificed at various times to test for the presence of virus and 
expression of the bFGF gene. At minimum, rats are examined at days 4, 7, 
14, one month, and two months. If these initial studies are positive, the 
studies will be extended to longer times. 
Rats are killed by CO.sub.2 inhalation and then perfused intravascularly 
with phosphate buffered saline containing 2% paraformaldehyde and 2.5% 
glutaraldehyde. The eyes are bisected along the vertical meridian, 
embedded in epoxy resin, sectioned (1 .mu.m thick) and stained with 
toluidine blue. Sections are cut along the vertical meridian and contain 
all of the retina extending from the ora serrata in the inferior 
hemisphere passing through the optic nerve head. To insure that oblique 
sections are not analyzed, the alignment of the rod outer segments are 
examined to confirm that they lie in the plane of the section. Slightly 
oblique sections show tangential fragments of the outer segments and are 
not used. 
Previous studies have shown that changes in retinal cell number 
(degeneration) result in thinning of the affected cell layer. LaVail et 
al., Proc. Nat'l Acad. Sci. USA 89:11249 (1992); Michon et al., Invest. 
Ophthalmol. Vis. Sci. 32:280 (1991); Unoki and LaVail, Invest. Ophthalmol. 
Vis. Sci. 35:907 (1994); Zhang et al., Invest. Ophthalmol. Vis. Sci. 
35:3163 (1994); Olsson et al., Neuron 815-830 (1992). Retinal degeneration 
will be quantified by measuring the mean thickness of (1) retina from the 
outer limiting membrane to the inner limiting membrane, (2) the border of 
the inner plexiform layer to the inner limiting membrane, (3) the inner 
nuclear layer, and (4) the outer nuclear layer. These are essentially the 
methods used by Unoki and LaVail, Invest. Ophthalmol. Vis. Sci. 35:907 
(1994), and Zhang et al., Invest. Ophthalmol. Vis. Sci. 35:3163 (1994). If 
necessary, eye tissues may also be examined by transmission electron 
microscopy to detail cell structure and preservation, as described 
previously. Brandt et al., J. Invert. Pathol. 32:12 (1978). 
In a similar manner, studies can be extended to other animal models of 
disease to determine if the treatment will be broadly applicable. Suitable 
models include the Royal College of Surgeons Rat, the rd mouse, and the 
rds mouse. 
All references discussed supra are hereby incorporated by reference. 
Although the foregoing refers to particular preferred embodiments, it will 
be understood that the present invention is not so limited. It will occur 
to those of ordinary skill in the art that various modifications may be 
made to the disclosed embodiments and that such modifications are intended 
to be within the scope of the present invention, which is defined by the 
following claims.