EP1082140A1

EP1082140A1 - Gene expression method

Info

Publication number: EP1082140A1
Application number: EP99925911A
Authority: EP
Inventors: Joseph C. Glorioso; Darren P. Wolfe; William F. Goins
Original assignee: University of Pittsburgh
Current assignee: University of Pittsburgh
Priority date: 1998-05-27
Filing date: 1999-05-27
Publication date: 2001-03-14
Also published as: WO1999061067A1; WO1999061067A8; AU4210399A; WO1999061067B1

Abstract

The present invention provides a method of delivering a factor to non-neural tissue and a method of treating neuropathy. The method involves introducing an expression cassette including a nucleic acid for expression involved in the production of the factor into a neural cell, within which the nucleic acid for expression is expressed, and from which the factor is therefore secreted. The method also provides a method of promoting long-term gene expression in vivo, by employing a herpesvirus to deliver a transgene to a desired tissue of a host animal. The method also permits repeat administration of the transgene.

Description

GENE EXPRESSION METHOD

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of expressing a gene.

BACKGROUND OF THE INVENTION

Gene transfer technology has wide-ranging utility in a number of applications relating to biological research and the treatment of disease. The technology typically involves the transfer of genetic material, typically a gene, to a cell (the host cell). Within the host cell, the gene is expressed to produce a biologically important factor within the cell.

Therapeutic products are typically delivered to a given tissue type by infecting that tissue. For example, to treat cystic fibrosis, the gene the cystic fibrosis transmembrane conductance regulator is transferred to cells in the diseased lung epithelium (Rosenfeld et al., Science, 252, 431-34 (1991); Crystal et al., Nature Genetics, 8, 42-51 (1994); Drumm et al., Cell, 52, 1227-33 (1990)). Similarly, for treatment of cancer, the gene encoding the herpesvirus thymidine kinase (tk) can be expressed within tumor cells, which are then exposed to prodrugs to kill transfected cells along with cells in the immediate vicinity (see Moolten, Cancer Res., 46, 5276 (1986); Moolten et al., J. Nat. Cancer Inst., 82, 297 (1990); Faulds et al., Drugs, 39, 597 (1990)). Thus, typically the desired factors are produced within the cells or tissues to be affected.

In many applications, the requirement to deliver genetic vectors to the tissues to be affected imposes unwelcome consequences. For example, it has been proposed that the delivery of the interleukin-1 receptor antagonist (IL-lra) to the intestinal epithelium might pose an effective treatment to inflammatory bowel disease (Walter et al., J. Controlled Release, 46, 75-87 (1997)). This approach involves the infection of intestinal epithelium with adenoviral vectors harboring the IL-lra gene. A noted drawback is that "[s]ince the intestinal epithelium renews most of its cell population rapidly (2-4 days), repeated application of the vector would be required" (Id.).

It has been suggested that gene transfer applications can deliver some products systemically. For example, a recombinant adenovirus has been employed to deliver erythropoeitin systemically following intramuscular injection (Kay et al., Proc. Nat. Acad. Sci. (USA), 91, 11557-61 (1994); Setoguchi et al., Blood, 8, 2946-53 (1994)). However, such protocols are plagued by a host immune response against virally-transduced cells (see, e.g., Yang et al., J. Virol, 69, 2008- 15 (1995)). This inflammatory response can interfere with the delivery of the desired factor or result in complications secondary to the transfer of genetic material. Indeed, in some applications, this host immune response virtually eliminates virally-transduced cells (see, e.g., Yang et al., supra; Gilgenkrantz et al., Hum. Gene. Ther., 6, 1265-74 (1995); Yang et al, J. Virol, 70, 7209-12 (1996)).

In view of the foregoing problems, there exists a need for an improved method of delivering a biologically-important factor to desired cells using gene- transfer technology, specifically while minimizing the host immune response and invasiveness of the procedure.

SUMMARY OF THE INVENTION

The present invention provides a method of delivering a factor to non- neural tissue and a method of treating neuropathy. The method involves introducing an expression cassette including a nucleic acid for expression involved in the production of the factor into a neural cell, within which the nucleic acid for expression is expressed, and from which the factor is therefore secreted. The method also provides a method of promoting long-term gene expression in vivo, by employing a herpesvirus to deliver a transgene to a desired tissue of a host animal. The method also permits repeat administration of the transgene. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.

DETAILED DESCRIPTION For purposes of describing the invention, the following definitions are used:

A cassette is a discrete and identifiable polynucleotide sequence. Thus a cassette can comprise a vector or it can comprise a component of a vector. An example of a cassette is an expression cassette, which is a cassette minimally comprising a nucleic acid for expression operably linked to a promoter sequence for driving the expression of the nucleic acid. In this context, a promoter is a nucleic acid sequence sufficient to drive the expression at appreciable levels of a second nucleic acid sequence to which it is operably linked. A nucleic acid for expression is a nucleic acid sequence corresponding to (i.e., encoding) a mature RNA species. Within an expression cassette, the nucleic acid for expression is operably linked to the promoter in that they are functionally associated such that an event at the promoter (e.g., binding cellular transcription factors) can precipitate a response from the nucleic acid for expression. Thus, in the context of the present inventive method, the nucleic acid for expression is expressed if it is transcribed within the cell.

Mutation is broadly defined to mean any change from a native nucleic acid sequence. A mutation can be the deletion of at least one nucleotide; conversely, a mutation also can be the insertion of one or more non-native nucleotides. Of course, native sequences can be deleted and non-native sequences inserted to effect a replacement mutation. A mutation is inactivating if it serves to impair or obliterate the function of a cassette in whole or in part. A deficiency is an inactivating mutation within an expression cassette (e.g., such a mutation within the nucleic acid for expression or within the regulatory elements).

In one embodiment, the method involves introducing an expression cassette including a nucleic acid for expression whose product is involved in the production of the factor into a neural cell. Within the cell, the nucleic acid f is expressed, and the factor is produced within and secreted from the neural cell. The inventive method can be employed to deliver any desired factor to the non-neural tissue, provided the factor can be produced intracellularly within the nerve cell. Thus, for example, the factor can be an enzymatic product, a protein, a glycoprotein, a proteoglycan, an aggregate of proteins, a virus, etc. The neural cells to which the nucleic acid for expression is transferred can be within either the central nervous system or the peripheral nervous system. Moreover, the neural cells can be of any type (e.g., unmyelated C-fibers, peripheral motor neurons, ganglion cells, cells of the enteric nervous system, etc.). Because of the unique and varied scope of neuroanatomy, the method can be employed to target the factor broadly or discretely within desired tissue. As all tissue is innervated by neurons, the non-neural tissue can be of any type in which it is desired to deliver the factor. Thus, for example, the cell can be within epidermal tissue, dermal tissue, tissue of the digestive organs (e.g., cells of the esophagus, stomach, intestines, colon, etc., or their related glands), smooth muscle cells, such as vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, lung cells, hepatocytes, lymphocytes, endothelial cells, sclerocytes, kidney cells, glandular cells (e.g., those in the thymus, ovaries, testicles, pancreas, adrenals, pituitary, etc.), tumor cells, cells in connective tissue (e.g., blood, chondrocytes, keratinocytes, cells of the meninges, etc.), and other cells of interest. Where the neural cell communicates with the non-neural tissue, the factor can be secreted from the nerve terminals directly into the non-neural tissue of interest. To effect such targeted delivery of the factor, the expression cassette is transferred only to discrete nerve cell populations. However, it is known in the art that nerve cell populations typically are isolated, particularly in the periphery. Thus, to target a discrete tissue type, the expression cassette can be transferred, for example, to nerve cells within a ganglion servicing the non-neural tissue. For systemic delivery, the neural cell can secrete the factor into tissue able to adsorb the factor into the general circulation. Thus, for example, systemic delivery can be achieved by producing the factor in enteric nerves such that it is secreted into the basal epithelium (brush border) lining the intestinal lumen or secreted into a capillary bed (such as between the pituitary and hypothalamus). Alternatively, a factor able to cross the "blood-brain" barrier can be secreted into the cerebrospinal fluid, ultimately to be adsorbed into the general circulation.

The inventive method involves the introduction of an expression cassette into a cell (e.g., the neural cell). As mentioned above, the expression cassette minimally includes a promoter, and, in the context of the present invention, the promoter must be able to drive the expression of the nucleic acid for expression within a neural cell. Examples of suitable promoters include prokaryotic promoters (e.g., bacterial promoters) and viral promoters (e.g., retroviral ITRs, LTRs, immediate early viral promoters (IEp) (such as heφesvirus IEp (e.g., ICP4- IEp and ICPO-IEp) and cytomegalovirus (CMV) IEp), and other viral promoters (e.g., late viral promoters, latency-active promoters (LAPs), Rous Sarcoma Virus (RSV) promoters, and Murine Leukemia Virus (MLV) promoters)). Other suitable promoters are eukaryotic promoters, such as enhancers (e.g., the rabbit β-globin regulatory elements), constitutively active promoters (e.g., the β-actin promoter, etc.), signal specific promoters (e.g., inducible and/or repressible promoters, such as a promoter responsive to TNF or RU486, the metallothionine promoter, etc.), and neural-specific promoters.

While, as mentioned, any promoter active within a neural cell can be employed, to effectuate the inventive method, the present invention provides a LAP2-driven expression cassette. A LAP2 promoter is a sequence which, when within a herpesviral vector, drives gene expression within a neural cell at times after the native herpes immediate early promoters have been silenced. Nucleic acid sequences from which such a LAP2 promoter can be derived are known in the art (Goins et al., J. Virol, 68(4), 2239-52 (1994)). However, sequences can vary among different strains of HSV and between the genomes of HSV-1, and other herpesviruses (e.g., HSV-2, see McGoech et al., J. Gen. Virol, 72, 3057-75 (1991) for a comparison of HSV-1 and HSV-2). This natural scope of allelic variation is included within the scope of the invention. Moreover, the LAP2 promoter has been extensively characterized by deletion, insertion, and substitution mutagenesis (see, e.g., Goins et al., supra; French et al., Mol Cell Biol, 16(10), 5339-99 (1996); see also International Patent Application WO 96/27672), and the present invention includes such recombinant promoters having LAP2 activity. Thus, a LAP2 promoter can, in some contexts, be or comprise an active fragment of a native LAP2 promoter (e.g., a deletion mutant). Such promoters can also include (e.g., non LAP2) sequences, such as an insertion mutant including exogenous DNA flanked by LAP2-derived sequences or even including LAP1 sequences. Additionally and alternatively, the LAP2 promoter can be a sequence derived from a LAP2 promoter that includes one or more point mutations or any other naturally occurring LAP2 region.

Regardless of the promoter employed, the expression cassette also minimally includes a nucleic acid for expression. While many nucleic acids for expression correspond to messenger RNA, thus encoding proteins, other gene products are non-translated RNA species (e.g., rRNA, antisense RNA, ribozymes, etc.). Generally, a nucleic acid for expression encodes a protein involved in the production of the factor to be secreted from the neural cell. For example, the nucleic acid for expression can encode an enzyme for synthesizing the factor (e.g., an enzyme catalyzing any step in the process of synthesizing the factor). Of course, where the factor to be secreted from the neural cell is a protein, the nucleic acid for expression can encode that protein or a derivative of the protein (e.g., a fusion of the protein and a secretion leader sequence, or a fusion of the protein and a proteinatious means for passing the blood-brain barrier).

The inventive method is broadly applicable and not limited to any species of nucleic acid for expression. Of course, where it is desired to employ gene transfer technology to supply a given factor to a tissue, the sequence of a protein involved in the production of the factor will be known in the art. Thus, the nucleic acid for expression can encode a cytokine (e.g., tumor necrosis factor (TNF), TGF- α, TGF-β, interleukins (IL) such as IL-1, IL-2, IL-3, etc., GM-CSF, G-CSF, M- CSF, co-stimulatory factor B7, IL-ira, etc.), a globin molecule (e.g., hemoglobin, β-globin, etc.), a trophic factor such as a neurotrophic factor (e.g., nerve growth factor (NGF), ciliary neurotrophic factor, brain derived neurotrophic factor, glial derived neurotrophic factor, neurotrophin-3, etc.) a somatic growth factor (e.g., human growth factor (HGF), adrenocorticotrophic factor (ACTH), platelet-derived growth factor (PDGF), epidermal growth factor (EGF)) or other trophic factor (e.g., thyroid-stimulating hormone (THS), follicle-stimulating hormone (FHS), leutinizing hormone (LH), prolactin, somatomammotropin, etc.), another peptide hormone (e.g., insulin, glucagon, parathormone, erythropeitin, antidiuretic hormone (ADH)), a peptide neurotransmitter (e.g., pituitary peptides mentioned above, enkephalins (e.g., proenkephalin, preproenkephalin, etc.), substance P, cholecystokinin, vasoactive intestinal polypeptide (VIP), neurotensin, angiotensins, bradykinin, camosine, bombesin, etc.), an immunoglobulin, a blood clotting factor (e.g., Factor VIII, Factor IX, etc.), other enzymes (e.g., phenylaniline hydroxylase, αl-antitrypsin, lacZ, etc.), or other protein of interest.

Where the nucleic acid for expression encodes a neuroactive factorm it need not be delivered to non-neural tissue. Such neurotrophic factor can be an enzyme or RNA acting within a neuron, or a secreted factor (e.g., neurotrophic factors or neurotransmitters) acting on neurons. In this embodiment, the invention provides a method of treating neuropathy by introducing into a neural cell an expression cassette comprising a nucleic acid for expression involved in the production of the neuroactive factor. Within the cell, the neuroactive factor is produced to treat the neuropathy. For example, the method can be employed to treat certain symptoms of diabetes where the factor is a neurotrophic factor; alternatively, the method can be employed to treat pain, for example where the factor is an enkephalin. Production of the factor within the nerve cell treats the neuropathy by reducing or attenuating the neuropathological symptoms. While in some applications, the method can lead to complete remission of such symptoms, any degree of relief from such effects would be considered sufficient treatment of the condition by those of skill in the art.

In accordance with the inventive method, within the expression cassette, the nucleic acid for expression and the promoter are operably linked such that the promoter is able to drive the expression of the nucleic acid. As long as this operable linkage is maintained, the expression cassette can include more than one nucleic acid for expression, such as multiple genes separated by ribosome entry sites. Furthermore, the expression cassette optionally can include other elements, such as polyadenylation sequences, transcriptional regulatory elements (e.g., enhancers, silencers, etc.), or other sequences. Where the encoded product of the nucleic acid for expression is the factor to be secreted from the neural cell, preferably the expression cassette includes a leader sequence for promoting the secretion of the translated protein as a part of the nucleic acid for expression (See, e.g., Suter et al, EMBO, J., 10, 2395-2400 (1991); Beutler et al., J. Neurochem., 64, 475-81 (1995)); of course, some proteins which are secreted from cells naturally include such sequences, while others can be expressed as a fusion protein. Where the factor is to pass across the blood-brain barrier, it desirably includes a means for passing the blood-brain barrier. Thus, for example, the factor can be a fusion protein including a portion of the transferrin molecule or a portion of an antibody recognizing the transferrin receptor (see, e.g., McGrath et al., J. Neurosci. Res., 47, 123-33 (1997); Friden et al., Science, 259, 373-77 (1993); Friden et al, Proc. Nat. Acad. Sci. (USA), 88, 4771-75 (1991)).

The expression cassette must be introduced into the neural cell in a manner suitable for the cell to express the gene(s) contained therein. Any suitable vector can be so employed, many of which are known in the art. Examples of such vectors include naked DNA vectors (such as ohgonucleotides or plasmids), viral vectors such as adeno-associated viral vectors (Berns et al., Ann. N. Y. Acad. Sci., 772, 95-104 (1995)), adenoviral vectors (Bain et al., Gene Therapy, 1, S68 (1994)), heφesvirus vectors (Fink et al., Ann. Rev. Neurosci., 19, 265-87 (1996)), packaged amplicons (Federoff et al., Proc. Nat. Acad. Sci. USA, 89, 1636-40 (1992)), pappiloma virus vectors, picornavirus vectors, polyoma virus vectors, retroviral vectors, SV40 viral vectors, vaccinia virus vectors, and other vectors. In addition to the expression cassette of interest, the vector can also include other genetic elements, such as, for example, cassettes for expressing a selectable marker (e.g., β-gal or a marker conferring resistance to a toxin), a pharmacologically active protein, a transcription factor, or other biologically active substance.

Once a given type of vector is selected, its genome must be manipulated for use as a background vector, after which it must be engineered to incoφorate exogenous polynucleotides. Methods for manipulating the genomes of vectors are well known in the art (see e.g., Sambrook et al., supra)) and include direct cloning. site specific recombination using recombinases, such as the flp recombinase or the cre-lox recombinase system (reviewed in Kilby et al. Trends Genet., 9, 413-21 (1993)), homologous recombination, and other suitable methods of constructing a recombinant vector (see, e.g., International Patent Application WO 98/51809). In this manner, the expression cassette can be inserted into any desirable locus of the vector. Such insertions can disrupt one or more genes present in the native vector, if desired, or the expression cassette can be inserted between genetic elements to minimize perturbation of the vector genome. Indeed, as one type of LAP2 sequence is already present in heφesviral vectors, an expression cassette including a LAP2 promoter can comprise a native heφesviral LAP2 promoter operably linked to the nucleic acid of interest.

Where the vector is a viral vector, preferably the vector is replication incompetent in neural cells. Thus, for example, an adenoviral vector preferably has an inactivating mutation in at least the El A region, and more preferably in region El in combination with inactivating mutations in region E2 (i.e., E2A, E2B, or both E2A and E2B), and/or E4 (see, e.g., International Patent Application WO 95/34671). Where the viral vector is an AAV vector, it can be deficient in AAV genes encoding proteins associated with DNA or RNA synthesis or processing or any step of viral replication including capsid formation (see, e.g., U.S. Patents 4,797,368, 5,354,768, 5,474,935, 5,436,146, and 5,681,731). Where the virus is a retroviral vector, for example, the retroviral cis-acting encapsidation sequence (E) essential for virus production in helper cells can be deleted upon reverse transcription in the neural cell to prevent subsequent spread of the virus (see, e.g., U.S. Patent 5,714,353). Where the virus is a heφesvirus, inactivation of the ICP4 locus and/or the ICP27 genes renders the virus replication incompetent in any cell not complementing the proteins (see, e.g., U.S. Patent 5,658,724, see also DeLuca et al., J. Virol, 56, 558-70 (1985); Samaniego et al., J. Virol 69(9), 5705-15 (1996)).

While any vector can be employed within the context of the present inventive method, preferably the vector is a heφesviral vector because such vectors are naturally neurotropic. Moreover, within neural cells, a heφesvirus enters a latent state, even in the presence of specific host immune response (Rock, Sem. Virol, 4, 157-65 (1993)). During this latent phase, the virus persists in an episomal form for the life of its host (Mellerick and Fraser, Virology, 158, 265-75, (1987); Rock and Fraser, J. Virol, 55, 849-52, (1985)), neither interfering with cellular function nor inducing autoimmune response (Ramakrishnan et al., J. Virol, 68, 1864-70 (1994); Fruh et al., Nature, 375, 415 (1995)). During the latent state, most viral promoters become progressively silenced as, indeed, do most exogenous promoters. The notable exception to this progressive silencing, however, is the heφesviral LAP promoters (e.g., LAP1, and LAP2). Thus, where the expression cassette includes a LAP2 promoter, such as those described above, the cassette is most preferably within a heφesviral vector, so that the conditions of viral latency can be established sufficient to activate expression from the LAP2 promoter. Thus, the invention provides a method for long-term expression of the nucleic acid for expression within the host cells. In addition to permitting long- term expression, through employing a heφesviral vector, the method permits repeat administration of the factor, which, as mentioned, is not possible with many other viral vector systems. Thus, following the initial administration of the vector, the method can involve "boosting" the initial inoculation by again introducing into the animal a second replication defective heφesviral vector comprising the non- heφes nucleic acid for expression and a promoter operatively linked to the nucleic acid. The second vector can be identical to the vector initially introduced, or it can be different. Preferably, the nucleic acid is expressed for at least about 50, such as at least about 75 days; however, the method can permit gene expression for at least about 100 days (such as at least about 125 days), or even longer (e.g., at least about 150 days or even at least about 300 days).

Where the vector is a heφesviral vector, aside from the inactivating ICP4 and/or ICP27 mutations mentioned above, preferably its genome is further modified (e.g., to render the vector replication deficient). Preferred additional mutations affect cassettes having the genes encoding tyrosine kinase (tk), ribunucleotide reductase (RR), γ34.5, Us3, and the transactivation function of VP16, ICP0. ICP22, and/or UL41. Desirably, such mutations are inactivating mutations. Disruption of the RR, γ34.5, Us3, ICP22. or the activation domain of VP16 genes renders the virus conditionally replication competent (Field et al., J. Hygiene, 81, 267-77 (1978); Cameron et al., J. Gen. Virol, 69, 2607-12 (1988); Fink et al., Hum. Gene Ther., 3, 11-19, (1992); Jamieson et al., J. Gen. Virol, 76, 1417-31 (1995); Chou et al., Science, 250, 1262-66 (1990); Sears et al, J. Virol, 55, 338-46 (1985)).

Aside from affecting replication, disruption of native heφesviral genes also can confer other advantageous phenotypes for use in the present invention. For example, joint inactivation of ICP4, ICP22, and ICP27 substantially reduces vector toxicity and reduces background expression of early and late genes to virtually undetectable levels. Inactivation of ICP0 greatly reduces expression from promoters other than the LAP2 promoters. Inactivation of the UL41 gene prevents the heφesviral host shut-off mechanism from properly functioning, thus accentuating expression of foreign expression cassettes within the infected cell. A heφesviral vector deficient for ICP47 helps to contain the infection within neural cells. Deleting the ICP47 gene greatly increases immune presentation of infected cells, other than nerve cells, as the ICP47 gene product normally interferes with MHC-I presentation of processed antigen on the cell (York et al., Cell, 77, 525-35 (1994)). Within neural cells, however, the expression of this gene is attenuated, and so the presence of the gene in the vector is of marginal importance. Moreover, many neural cells are immunologically privileged and are generally not subject to immune attack despite the presence of some ICP47 gene product. A vector harboring an expression cassette is introduced into the target cell by any means appropriate for the vector employed. Many such methods are well- known in the art (Sambrook et al.. supra; see also Watson et al.. Recombinant DNA, Chapter 12, 2d edition, Scientific American Books (1992)). Thus, plasmids are transferred by methods such as calcium phosphate precipitation, electroporation, liposome-mediated transfection, microinjection, viral capsid- mediated transfer, polybrene-mediated transfer, protoplast fusion, etc. Viral vectors are best transferred into the cells by infecting them; however, the mode of infection can vary depending on the virus. For example, most viruses able to infect neural tissue should be exposed directly to the desired neural cells (e.g., cells within a discrete region of the central nervous system, cells within a desired ganglion, etc.). Some viruses, such as HSV, display neurotropism, and this property can be exploited within the context of the inventive method. For example, heφesviral vectors can be delivered to nerve cells by infecting tissue into which the cells project or innervate (such as the non-neural tissue to which the factor is delivered in accordance with the inventive method). When exposed to such tissue, heφesviral vectors will preferentially infect neural cells in connection with such tissue (see, e.g., Gesser et al., J. Virol, 77(5), 4103-06 (1997); Gesser et al., Proc. Nat. Acad. Sci. (USA), 73(6), 880-89 (1995); Gesser et al, J. Virol, 70(6), 4097-4102 (1996)).

The mode of delivery also depends on the location of the cells at the time the vector is transferred. Thus, where the tissue is a peripheral ganglion or nucleus within the central nervous system, a bolus of a composition containing the vector can be injected into the coφus of the ganglion or nucleus. Where the enteric nervous system is targeted, the vector can be within a composition suitable for oral administration (such as a solution or pill, see, e.g., Gesser et al., 1995, 1996. and 1997, supra). The dose administered to an animal, particularly a human, in the context of the present invention will vary with the nucleic acid for expression of interest, the vector, the composition employed, the method of administration, and the particular site and organism being treated. However, the dose should be sufficient to effect the expression of the nucleic acid for expression of interest. One of skill in the art is well equipped to optimize these parameters to adapt the inventive method to a desired end use. Such perfection is routine within the art and requires no undue experimentation.

EXAMPLES

While it is believed that one of skill in the art is fully able to practice the invention after reading the foregoing description, the following examples further illustrate some of its features. In particular, they demonstrate that the introduction of an expression cassette including a nucleic acid for expression whose product is involved in the production of a given factor into a neural cell can effectively deliver the factor to non-neural tissue. Moreover, they demonstrate that the use of a heφesvirus to express a given nucleic acid can result in long-term expression of the nucleic acid. As these examples are included for purely illustrative pmposes, they should not be construed to limit the scope of the invention in any respect.

Many procedures, such as Southern blots, vector construction (including DNA extraction, isolation, restriction digestion, ligation, etc.), cell culture, transfection and infection of cells, protein assays (such as ELIS A, Western blotting, and β-galactosidase assays) are techniques routinely performed by those of skill in the art (see generally Sambrook et al., Molecular Cloning, supra)). However, some reagents employed in the following Examples deserve specific description.

The pBS-lox shuttle plasmid and dl20/tk-::lox virus system for cloning into the HSV tk locus (Rasty et al., Methods and Molecular Genetics, 7, 114-30 (1995)) was employed to construct the vectors described below. KOS is a wild-type HSV virus used as a control. The heφesviral KHN vector has an exogenous expression cassette including the lacZ gene under the control of the HSV gC promoter and linked to the SV40 polyadenylation sequence and a second expression cassette including the β-NGF gene operatively linked to the HCMV immediate early promoter and the SV40 polyadenylation sequence. Both cassettes are present within a larger cassette inserted into (and destroying) the native HSV tk locus. The cassette was created and cloned into the shuttle vector pBS-lox as a BamHI fragment to create a cloning plasmid (pBS-lox-KHN). Subsequently, the KHN virus was constructed using cre-lox recombination between dl20/tk-: :lox viral DNA and pBS-lox-KHN. The virus was plaqued and the genotype of the virus was confirmed by Southern hybridization of blue (lacZ-positive) plaques.

The heφesviral SLN vector has an exogenous expression cassette including the lacZ gene under the control of the HSV gC promoter and linked to the SV40 polyadenylation sequence and a second expression cassette including the β-NGF gene operatively linked to a LAP2 promoter and the S V40 polyadenylation sequence. Both cassettes are present within a larger cassette inserted into (and destroying) the native HSV tk locus. The virus was created similarly to KHN. The heφesviral SHN vector has an exogenous expression cassette including the lacZ gene under the control of the HSV gC promoter and linked to the SV40 polyadenylation sequence and a second expression cassette including the β-NGF gene operatively linked to an HCMV immediate early promoter and the SV40 polyadenylation sequence. The virus was created similarly to KHN.

The heφesviral SHZ vector (used as a control) has an exogenous expression cassette including the lacZ gene under the control of the HCMV promoter and linked to the SV40 polyadenylation sequence. The virus was created similarly to KHN.

EXAMPLE 1

This example demonstrates that the introduction of an expression cassette including a nucleic acid for expression whose product is involved in the production of a given factor into a neural cell can effectively deliver the factor to non-neural tissue.

BALB/C mice were inoculated in the intestinal track with about 5x10⁶ pfu of each of the viral strains KOS, SLN, and KHN. At two day intervals over a three week period, blood was withdrawn from each infected mouse, and the concentration of NGF was measured in each sample of withdrawn blood using a standard ELISA.

The results indicated that the concentration of NGF was not elevated appreciably upon inoculation with the KOS virus. This group of mice was not followed after five days, because they died from the heφes infection. Infection with the KHN virus effected a steady increase in NGF concentration for about a one week period, to a maximum of about 600 pg/ml, after which the plasma NGF concentration steadily declined to near uninfected levels. Infection with the SLN virus produced an elevation in plasma NGF concentration only after several days. However plasma NGF concentration from SLN-infected mice remained elevated (about 1200 g/ml) for the duration of the study.

That the activity HCMV-driven NGF cassette in the KHN vector declined after about one week post infection indicates that the vector primarily infects neural cells, as HCMV promoters are known to inactivate upon the establishment of latency. Moreover, the continued elevation of plasma NGF levels in the SLN- infected mice is inconsistent with a primarily epithelial infection (Walter et al., supra). These results are consistent with the observations that enteric infection of heφesviral infection is primarily neural (Gesser et al., 1995, supra, Gesser et al., 1996, supra, Gesser et al., 1997, supra). Together these results demonstrate that the introduction of an NGF expression cassette into neural cells can effectively deliver NGF to non-neural tissue, and even systemically. EXAMPLE 2

This example demonstrates that the use of a heφesvirus to deliver an expression cassette including a nucleic acid for expression whose product is involved in the production of a given factor can result in stable long-term expression of a desired nucleic acid for expression.

NZW rabbits were inoculated in one knee with about lxlO⁸ pfu the SHN, and SHZ (which substitutes the E. coli lacZ gene for NGF). At various intervals over a 300-day period, blood was withdrawn from each infected rabbit, and the concentration of NGF was measured in each sample of withdrawn blood using a standard ELISA. The experiments were run in duplicate.

Infection with the SLN virus produced an elevation in plasma NGF concentration after about one week, which lasted throughout the duration of the experiment (between about 5000 and about 8000 pg/ml). However plasma NGF concentration from SHN-infected rabbits rose to between about 20,000 and about 30,000 pg/ml, and gradually declined thereafter. At 300 days post infection, however, the SHN-infected animals still exhibited elevated levels of NGF. Rabbits infected with control (SHZ) viruses exhibited no appreciable elevation in serum NGF concentration. The results indicate that the use of a heφesvirus can result in stable long-term expression of a desired transgene.

EXAMPLE 3 This example demonstrates that the use of a heφesvirus as a genetic vector can permit repeat dosing of an expression cassette including a nucleic acid for expression of interest. After about 300 days post infection, the SHN-infected rabbits described in

Example 2 were inoculated in the same knee with about lxl 0⁸ pfu SHN. At various intervals, blood was withdrawn and assayed as described in Example 1. After about one week, plasma concentration of NGF climbed again to about 30,000 pg/ml, and gradually declined thereafter. The results demonstrate that the use of heφesviral vectors permit repeat dosing in gene transfer applications.

EXAMPLE 4

This example demonstrates that the use of a heφesvirus to deliver an expression cassette including a nucleic acid for expression whose product is involved in the production of a given factor can result in stable long-term expression of a desired nucleic acid for expression. Rhesus Macaque monkeys were inoculated in one knee with about lxlO⁸ pfu the SHN, or SHZ. At intervals over a 40-day period, blood was withdrawn from each infected monkey, and the concentration of NGF was measured in each sample of withdrawn blood using a standard ELISA. The experiments were run in duplicate.

Infection with the SHN virus produced an elevation in plasma NGF concentration after about one week (to about 50 ng/ml), and at 40 days post infection, the animals still exhibited elevated NGF (about 40 ng/ml NGF). Monkeys infected with control (SHZ) viruses exhibited no appreciable elevation in serum NGF concentration. The results indicate that the use of a heφesvirus can result in stable long-term expression of a desired transgene.

EXAMPLE 5

This example demonstrates that the introduction of an expression cassette including a nucleic acid for expression whose product is involved in the production of a given factor into a neural cell can effectively deliver a biologically significant amount of the factor.

Lavage fluid withdrawn from the knees of the SLN-infected rabbits, as discussed in Examples 2, was exposed to primary DRG or PC 12 cells in culture. Fluid from uninfected animals was used as a control. The withdrawn fluid was used to substitute for plasma otherwise added to the cell culture media.

After 10 days, the cells exposed to the fluid from SLN-infected animals grew neurites and differentiated. In contrast, cells exposed to blood from uninfected animals exhibited no change in phenotype. The observed phenotypic change is consistent with the reported differentiation of such cells upon exposure to β-NGF, indicating that the animals produced a biologically significant amount of NGF following infection.

EXAMPLE 6 This example demonstrates that the introduction of an expression cassette including a nucleic acid for expression whose product is involved in the production of a neuroactive factor into a neural cell can treat neuropathy.

A population of albino rats was induced to become diabetic, which results in progressive neuropathy. Such animals develop abnormal frequency of micturition as a result of damage to the sensory nerves serving the bladder. The bladders of one subpopulation of such rats were inoculated with the SLN vectors, while a control group was inoculated with SLZ or were sham inoculated. After 10 days, the animals were observed. The control groups possessed diabetic micturitive behavior, while those receiving the NGF-expressing virus exhibited normal micturitive behavior. These results indicate that the viruses infected the nerves servicing the bladder and that the nerves receiving the SLN vector expressed NGF. Moreover, the results indicate that the expression of NGF within such cells was sufficient to treat the neuropathy in such nerves.

All of the references cited herein, including patents, patent applications, and publications, are hereby incoφorated in their entireties by reference. The invention has been described with an emphasis upon preferred embodiments and illustrative examples. However, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Moreover, as the foregoing examples are included for purely illustrative puφoses, they should not be construed to limit the scope of the invention in any respect. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A method of delivering a factor to non-neural tissue, said method comprising introducing into a neural cell an expression cassette comprising a nucleic acid for expression involved in the production of said factor such that said factor is produced and secreted from said cell.

2. The method of claim 1, wherein said neural cell communicates with said non-neural tissue such that said factor is secreted into said non-neural tissue.

3. The method of claim 1 or 2, wherein said factor is disseminated systemically.

4. The method of claim 1, wherein said factor is first secreted into cerebrospinal fluid and then crosses the blood-brain barrier.

5. The method of any of claims 1-4, wherein said expression cassette is contained within a viral vector and is introduced into said neural cell by infecting said neural cell with said viral vector.

6. The method of claim 5, wherein said vector is a he╧åesviral vector.

7. A method of treating neuropathy, comprising introducing into a neural cell an expression cassette comprising a nucleic acid for expression involved in the production of a neuroactive factor such that said factor is produced within said cell to treat said neuropathy.

8. The method of any of claims 5-7, wherein said vector has an inactivating mutation in at least one viral locus essential for viral replication.

9. The method of any of claims 1-8, wherein said factor is a protein, and said nucleic acid for expression encodes said protein.

10. The method of any of claims 1-8, wherein said nucleic acid for expression encodes an enzyme for synthesizing said factor.

11. The method of any of claims 1-10, wherein said neural cell is within the peripheral nervous system.

12. The method of any of claims 1-10, wherein said neural cell is within the central nervous system.

13. The method of any of claims 1-12, wherein said expression cassette contains a LAP2 promoter.

14. A method of expressing a nucleic acid for expression in vivo, which comprises introducing into a tissue of an animal a replication defective he╧åesviral vector comprising a non-he╧åes nucleic acid for expression and a promoter operatively linked to said nucleic acid for expression, whereby said nucleic acid for expression is expressed in vivo for at least about 40 days.

15. The method of claim 14, further comprising again introducing into said animal a second replication defective he╧åesviral vector comprising a non-he╧åes nucleic acid for expression and a promoter operatively linked to said nucleic acid for expression, whereby said nucleic acid for expression is again expressed in vivo for at least about 40 days.

16. The method of claim 14 or 15, wherein said nucleic acid for expression is expressed for at least about 100 days.

17. The method of any of claims 14-16, wherein said nucleic acid for expression is expressed for at least about 200 days.

18. The method of any of claims 14-17, wherein said nucleic acid for expression is expressed for at least about 300 days.

19. The method of any of claims 14-18, wherein said nucleic acid for expression encodes a protein.

20. The method of any of claims 14-19, wherein said promoter is a LAP2 promoter.