A persistent HSV gene delivery system

Abstract

The present invention relates to herpes simplex virus (HSV)-based vectors for delivering transgenes (e.g., therapeutic genes) that are more resistant to neutralization by the immune system, phagocytosis, and NK cells, as well as methods for their preparation and use to treat disorders and diseases (such as those associated with gene expression). In one embodiment, the HSV vectors are prepared by treatment with immune serum containing high levels of anti-HSV antibodies. The HSV vectors may include an extracellular CD47 domain inserted at the N-terminus of the glycoprotein to inhibit phagocytic activity, and the absence of gE to avoid NK cells.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 269,440, filed March 16, 2022, which is incorporated herein by reference. (Technical field)

[0002] The present invention relates to non-replicating herpes simplex virus (HSV)-based gene delivery vectors for delivering transgenes (e.g., therapeutic genes) that are more resistant to neutralization and phagocytosis by the immune system, as well as methods for their preparation and use to treat disorders and diseases, such as those associated with gene expression. [Background technology]

[0003] Gene therapy has many potential clinical applications for treating various diseases, such as genetic diseases, autoimmune diseases, and cancer. The success of gene therapy depends heavily on the availability of gene delivery vectors, such as viral vectors. An ideal gene delivery vector can efficiently deliver therapeutic genes to body tissues and ensure that the delivered therapeutic genes can remain in the tissues for a long time. However, the effectiveness of viral vectors is often reduced by various defense mechanisms of the host in the body.

[0004] For example, anti-HSV neutralizing antibodies bind to transduced viral particles, such as herpes simplex virus (HSV) particles. These antibodies can be either pre-existing due to the patient's previous exposure to the virus or newly arising from repeated administration of the virus during treatment. Neutralizing antibodies can reduce gene transfer efficiency by several mechanisms. First, they prevent the virus from infecting cells. Second, they can bind to transduced cells and lead to their destruction by the host's antibody-dependent cellular phagocytosis (ADCP) or by activation of the complement system of the membrane attack complex (MAC) (Huber et al., 2001; Tay, Wiehe, and Pollara, 2019; Xie, Jane-Wit, and Pober, 2020). Indeed, animal studies have shown that pre-existing humoral immunity is detrimental to HSV infectivity (Fu and Zhang, 2001).

[0005] Anti-HSV neutralizing antibodies are found to primarily target two of the virus-encoded glycoproteins: glycoprotein D (gD) and gB (Cairns et al., 2015, Cairns et al., 2014). Both gD and gB contain many neutralizing epitopes and present in different individuals in various orders of immunodominance hierarchy (Bender et al., 2007). Overall, these epitopes are well conserved among infected individuals and / or different species (Eing, Kuhn and Braun, 1989). Studies have also shown that HSV-2 is more difficult to neutralize than HSV-1 (Silke Heilingloh et al., 2020). The presence of strong epitopes presents as a major obstacle to systemic administration of HSV-based viruses, as neutralizing antibodies can easily recognize them and abolish their therapeutic activity.

[0006] Another important limiting factor that reduces the effectiveness of systemically delivered viral vectors is the host's mononuclear phagocyte system (MPS). It has been shown that after systemic delivery, viral particles can be rapidly removed by MPS (Ellermann-Eriksen, 2005; Hume, 2006; Van Strijp et al., 1989). Specifically, a study by Fulci et al. has shown that macrophage depletion can significantly improve the tissue distribution of HSV (Fulci et al., 2007).

[0007] Yet another important antiviral mechanism that may reduce the effectiveness of viral delivery by HSV is natural killer (NK) cells (Alvarez-Breckenridge et al., 2012a, Alvarez-Breckenridge et al., 2012b). The viral glycoprotein E (gE) is known to bind IgG (Dubin et al., 1994). Recent studies have shown that NK cells can recognize HSV or HSV-infected cells through the binding of the surface CD16 activating receptor with the Fc region of IgG bound to gE (Dai and Caligiuri, 2018).

[0008] One way to limit the effect of neutralizing antibodies on viral infectivity is by mutating each of the major epitopes. Indeed, recent studies have shown that mutating two of these epitopes from gD of HSV can actually abolish neutralization by the corresponding monoclonal antibody (mAb) (Tuzmen et al., 2020). However, both gB and gD of HSV-1 and HSV-2 are major targets of neutralizing antibodies, each of which contains nearly 12 neutralizing epitopes. In the context of a viral genome over 150 kb in length, using traditional methods to mutate each of these epitopes from the two glycoproteins is not practical. Furthermore, this approach addresses only one of the three major obstacles faced by systemic delivery of viral therapy (i.e., neutralizing antibodies), leaving MPS intact.

[0009] US Patent Publication No. 2012 / 0301506, and US Patent Nos. 8,986,672 and 10,039,796 disclose modified HSV-2 and their use in treating malignancies.

[0010] Fu et al., Oncotarget, 2018, 9(77):34543-34553, describes genetic engraftment of CD47 onto the membrane envelope of HSV to allow escape from MPS.

[0011] U.S. Patent Publication No. 2019 / 0267845 describes an HSV vector that does not express HSV genes in non-complementing cells and includes a genome that includes one or more transgenes, wherein the vector is capable of expressing the transgenes in the non-complementing cells for at least 28 days.

[0012] U.S. Patent Publication No. 2018 / 0148711 discloses an HSV-based genome editing vector. Verlengia et al., Sci Rep., 2017 May 4,7(1):1507, doi:10.1038 / s41598-017-01635-1, describe engineered HSV vectors for transgene expression in the central nervous system. There remains a need for persistent viral vectors suitable for gene therapy that are capable of resisting the restrictive effects from the host's innate and acquired antiviral immune mechanisms. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] US Patent Publication No. 2012 / 0301506 [Patent Document 2] U.S. Pat. No. 8,986,672 [Patent Document 3] U.S. Pat. No. 10,039,796 [Patent Document 4] US Patent Publication No. 2019 / 0267845 [Patent Document 5] US Patent Publication No. 2018 / 0148711 [Non-patent literature]

[0014] [Non-Patent Document 1] Fu et al.,Oncotarget,2018,9(77):34543-34553 [Non-Patent Document 2] Verlengia et al.,Sci Rep.,2017 May 4,7(1):1507,doi:10.1038 / s41598-017-01635-1 Summary of the Invention

[0015] The inventors have found that by passaging HSV vectors with immune serum with elevated levels of anti-HSV antibodies and / or by including an extracellular CD47 domain (e.g., an extracellular human CD47 domain) in the membrane envelope, HSV vectors can deliver transgenes more efficiently and the delivered transgenes can persist longer. Unexpectedly, it has been found that passaging CD47-containing viruses results in almost complete loss of gE from the virus particles. This allows the virus to evade NK cell-mediated antiviral mechanisms. It has also been found that inserting the extracellular domain of CD47 into the N-terminus of gC allows the virus to evade neutralization by anti-HSV antibodies even without passaging. The HSV vectors described herein can be non-oncolytic and non-toxic.

[0016] One embodiment of the present invention is a composition comprising a non-replicating herpes simplex virus (HSV)-based gene delivery vector, (i) the HSV-based vector comprises a transgene, and (ii) the HSV-based vector is prepared by passaging the HSV-based vector at least twice with immune serum having elevated levels of anti-HSV antibodies. In one embodiment, passaging in immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of the HSV-based vector. The vector also preferably comprises a promoter operably linked to the transgene. In one embodiment, the HSV-based vector is non-oncolytic. In another embodiment, the HSV-based vector is persistent.

[0017] The HSV-based vector can be HSV-1 or HSV-2. A preferred HSV-based vector is HSV-2.

[0018] The HSV-based vector can be an amplicon packaged into an HSV-1 particle or an HSV-2 particle.

[0019] In one embodiment, the HSV-based vector has a membrane envelope comprising glycoproteins, at least one of which comprises an extracellular CD47 domain inserted at the N-terminus of the glycoprotein. The glycoproteins may be selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L. In a preferred embodiment, the membrane envelope comprises glycoprotein C having an extracellular CD47 domain inserted at its N-terminus. The extracellular CD47 domain may be of mouse or human origin. The extracellular CD47 domain may comprise amino acids 19-141 of mouse CD47 (SEQ ID NO: 1). Alternatively, the extracellular CD47 domain may comprise amino acids 55-423 of human CD47 (SEQ ID NO: 3). The vector comprises a genome encoding the CD47 domain.

[0020] In another embodiment, the HSV-based vector is free or substantially free of gE.

[0021] The immune serum can be a mixture containing rat serum and human serum that have elevated levels of anti-HSV antibodies.

[0022] In one embodiment, the HSV-based vector is prepared by passaging the HSV-based vector at least two times (e.g., at least 5 or 7 times) in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by passaging at least two more times (e.g., at least 5, or 10, or 17, or 23 times) in the presence of a mixture of rat serum with elevated levels of anti-HSV antibodies and at least one human serum.

[0023] In one embodiment, the composition comprises cerebrospinal fluid.

[0024] Another embodiment is a non-replicating HSV-based vector comprising a transgene, the HSV-based vector having a membrane envelope comprising glycoproteins, at least one of which comprises an extracellular CD47 domain inserted at the N-terminus of the glycoprotein. In one embodiment, the HSV-based vector has a membrane envelope comprising glycoproteins, at least one of which comprises an extracellular CD47 domain inserted at the N-terminus of the glycoprotein. The glycoproteins may be selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L. In a preferred embodiment, the membrane envelope comprises glycoprotein C having an extracellular CD47 domain inserted at its N-terminus. The extracellular CD47 domain may comprise amino acids 19-141 of CD47 (SEQ ID NO: 1). Alternatively, the extracellular CD47 domain may comprise amino acids 55-423 of human CD47 (SEQ ID NO: 3). In another embodiment, the HSV-based vector is free or substantially free of gE. In a preferred embodiment, neutralizing epitopes on glycoprotein B and glycoprotein D of the HSV-based vector are mutated. The vector also preferably comprises a promoter operably linked to the transgene. In one embodiment, the HSV-based vector is non-oncolytic. In another embodiment, the HSV-based vector is persistent. The vector comprises a genome encoding an extracellular CD47 domain (which may be, for example, of murine or human origin).

[0025] Yet another embodiment is a method of preparing a composition comprising a non-replicating herpes simplex virus (HSV)-based vector (e.g., an HSV-based vector described herein), comprising passaging an HSV-based vector comprising a transgene at least twice with immune serum having elevated levels of anti-HSV antibodies. In one embodiment, the immune serum comprises a mammalian anti-HSV antibody. In another embodiment, the immune serum is a mixture comprising rat serum and human serum having elevated levels of anti-HSV antibodies. In yet another embodiment, the method comprises passaging the HSV-based vector at least twice (e.g., at least 5 times, or 7 times) in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by passaging at least once (e.g., at least 2 times, 5 times, or 10 times, or 17 times, or 23 times) in the presence of a mixture of rat serum and at least one human serum having elevated levels of anti-HSV antibodies.

[0026] Yet another embodiment is a method of treating a disease in a patient in need thereof, comprising administering to the patient a composition described herein or a non-replicating HSV-based vector described herein. The disease or disorder may be a neurological disease or disorder. In one embodiment, the disease or disorder is not a neoplastic disease or disorder (e.g., the disease or disorder is not cancer). The composition or HSV-based vector may be administered systemically, intramuscularly, intradermally, subcutaneously, or parenterally, or by intracerebroventricular or intraperitoneal injection. In one embodiment, the patient has been exposed to, or has been vaccinated against, or has HSV-1 and / or HSV-2.

[0027] Yet another embodiment is a method of vaccinating a patient against a malignant or infectious disease comprising administering to the patient a composition described herein or a non-replicating HSV-based vector described herein, in one embodiment, the transgene is one useful for vaccinating a patient. [Brief description of the drawings]

[0028] For a more complete understanding of the present invention, including its features and advantages, reference is now made to the detailed description of the invention taken in conjunction with the accompanying drawings.

[0029] [Figure 1] 1 is a bar graph showing the difference in sensitivity of trained or untrained FusOn-H2 to anti-HSV sera. 1×104 untrained FusOn-H2 or trained FusOn-SS9 were mixed with either human or rat anti-HSV-2 sera at a final concentration of 1:5. After 1 h incubation at 37° C., the solution was applied to Vero monolayers in 12-well plates. Plaques were challenged 48 h later with crystal violet staining. [Figure 2A] Schematic diagram of CD47 coding sequence insertion into HSV vector. It shows the insertion of EGFP-Luc gene cassette (transgene, which can be any therapeutic gene) and CMVp-HAtag-CD47ECD into the backbone of HSV vector (FusOn-H2) by homologous recombination, with the left flanking (LF) sequence and the 3' region of gC (from the transmembrane domain to polyA) as the right flanking sequence. Details of the individual components in the gene cassette are shown in the figure and labelled accordingly. Abbreviations are as follows: CMVp: Cytomegalovirus immediate early promoter, LTR: Rous sarcoma long terminal repeat containing the promoter region of this virus, GFP-Luc: EGFP-luciferase fusion gene, HA: HA tag, CD47: mouse CD47 extracellular domain, gC: complete gC coding region, LF: left flanking region of gC. Recombinant viruses were identified by expression of GFP and purified to homogeneity. [Figure 2B] Schematic diagram of FusOn-Luc construction strategy, showing that FusOn-Luc (as a control virus) containing only the transgene without CD47 was constructed in a similar manner, except that it does not contain the CD47 extracellular domain. [Diagram 3]Figure 1 is a chart showing a comparison of FusOn-CD47 and FusOn-Luc for their ability to transduce cells and replicate in the presence of phagocytes. This is a comparison of virus yield in the presence or absence of phagocytes. Vero cells in 12-well plates were infected with virus at 0.1 pfu / cell in the presence or absence of 200,000 splenocytes. Cells were harvested at 48 hours and virus yield was determined by plaque assay. *p<0.05 compared to other 3 wells. [Figure 4A] 1 is a chart showing the average photon readings (i.e., quantification of Luc transgene expression) from daily IVIS imaging measurements. This demonstrates that FusOn-CD47 is delivered more efficiently by the systemic delivery route (in this case, to tumor tissue) and the transgene persists longer than FusOn-Luc. Tumors were established in the right flank of Balb / c mice by subcutaneous implantation of CT26 cells. When tumors reached an approximate size of 8 mm in diameter, 2×106 pfu of either FusOn-CD47 or FusOn-Luc were administered systemically. [Figure 4B] Time-lapse images of transgene expression in a typical mouse after systemic delivery of FusOn-CD47. This shows that transgenes can be delivered by the systemic route more efficiently by FusOn-CD47 than by FusOn-Luc. Tumors were established on the right flank of Balb / c mice by subcutaneous implantation of CT26 cells. When tumors reached an approximate size of 8 mm in diameter, 2×106 pfu of either FusOn-CD47 or FusOn-Luc were administered systemically. Starting on day 2, animals were imaged daily for luciferase expression. [Diagram 5]1 is a chart showing the enhanced resistance of FusOn-CD47 to the neutralizing effect of anti-HSV serum after training. 1×104 untrained FusOn-CD47 or trained FusOn-CD47-SS24 (7 passages in the presence of rat serum and 17 passages in the presence of rat serum + 1 human serum) were mixed with either human or rat anti-HSV-2 serum at a final concentration of 1:5. After 1 hour incubation at 37° C., the solution was applied to Vero monolayers in 12-well plates. Plaques were challenged 48 hours later with crystal violet staining. [Figure 6] 1 is a chart showing that trained FusOn-CD47 acquired further resistance to anti-HSV immune serum. 5x103 untrained FusOn-CD47 or the same virus trained at different stages [sequential selection 24 (FusOn-CD47-SS24), sequential selection 49 (HR49, N2-N5 and N7-N8)] were mixed with 8 human serum mixtures at different dilutions. Trained FusOn-H2 (FusOn-SS9) was also included in this experiment. After 1 h incubation at 37°C, the solutions were applied to Vero monolayers in 12-well plates. Plaques were challenged 48 h later with crystal violet staining. [Figure 7] Figure 1 is a bar graph showing that CD47 insertion can enhance the ability of HSV vectors to evade neutralizing antibodies. 500 pfu of FusOn-CD47 and FusOn-Luc were used to infect Vero cells in 6-well plates after 1 h incubation at 37°C with anti-HSV-2 serum at the indicated dilutions (1:40 or 1:160). The same number of viruses was incubated with medium alone as a control. After 48 h, cells were stained with crystal violet to count viral plaques. The percentage of plaques was calculated by dividing the number of plaques in wells containing anti-HSV serum by the number of plaques from wells with the same virus without anti-HSV serum (control). * indicates p<0.05 compared to FusOn-Luc. [Figure 8]Figure 1 shows time-lapse images of transgene expression in a typical mouse several days after systemic delivery of FusOn-CD47, FusOn-Luc, or FusOn-SD. This shows that FusOn-CD47, but not FusOn-Luc, can be sufficiently delivered by the systemic route to mice with pre-existing anti-HSV immunity, and passaging FusOn-CD47 in serum containing anti-HSV antibodies (to generate FusOn-SD) further enhanced the ability of the virus to be delivered systemically in these mice. Balb / c mice were vaccinated with FusOn-H2 and then implanted with CT26 tumor cells in the right flank. Separate groups of mice received each of these three viruses at a dose of 1x107 pfu via the tail vein. Mice were imaged using an IVIS imager on the indicated days after virus injection. [Figure 9A] FIG. 1 is a bar graph showing enzyme-linked immunosorbent assay (ELISA) for expression of gE (by absorbance at 450 nm) in FusOn-H2, FusOn-SD, and PBS (negative control) after incubating the virus first with mouse anti-HSV-2 gE (1:1000 dilution) and then with HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). [Figure 9B] Western blot showing the absence of gE in FusOn-SD, but not in FusOn-H2. For Western blot detection, viruses were first incubated with mouse anti-HSV-2 gE (1:1000 dilution) and then with HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). [Figure 9C] FIG. 1 is a schematic diagram of the mechanism of gE-mediated NK cell recognition of HSV or HSV-infected cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Introduction Gene delivery vectors based on herpes simplex virus (HSV) have several unique features that make them attractive for certain clinical applications. For example, HSV genomes can persist in neural tissues for life, and thus HSV-based gene delivery vectors may be particularly useful for gene therapy of neurological disorders. However, because they are virus-based vectors, the host's antiviral immune mechanisms can often eliminate viral vectors, thus limiting their efficiency in gene delivery. Both innate and acquired antiviral immunity of the host can eliminate HSV-based vectors. The main innate antiviral immune components are macrophages and natural killer cells (NK cells), and the main acquired antiviral immune components are neutralizing antibodies. Here, we report the invention of a series of modifications of HSV-based gene delivery vectors that allow the virus to evade these antiviral mechanisms to enhance gene delivery efficiency. For phagocytosis by macrophages, the extracellular domain of CD47, a molecule containing a "don't eat me" signal, was inserted into the N-terminus of glycoprotein C (gC). This modification coats the viral vector with CD47, which prevents macrophages and other phagocytes from "eating" the viral vector. For neutralizing antibodies, the viral vector was subjected to a series of passages in the presence of anti-HSV immune serum. This not only selected vector candidates with the ability to withstand neutralizing antibodies, but also minimized the incorporation of gE into the viral particles. Since gE can bind to IgG, which can then be recognized by NK cells to kill cells transduced by HSV-based vectors, the absence of gE from the viral particles allows for more efficient gene transfer by allowing cells to escape from the NK cell-mediated killing effect.

[0031] Due to the inherent property of long-term persistence in nervous tissue, HSV-based gene delivery vectors are particularly useful for gene therapy applications in the treatment of neurological disorders.However, both innate and acquired antiviral mechanisms are major obstacles for HSV-based gene delivery vectors, including their gene delivery efficiency.The invention disclosed herein addresses these obstacles and allows viral vectors to be used more effectively for their clinical applications, illustratively for gene therapy.

[0032] definition The term "herpes simplex virus" or "HSV" as used herein refers to an encapsulated icosahedral double-stranded DNA virus that infects mammals, including humans. Wild-type HSV infects and replicates in both terminally differentiated non-dividing and dividing cells. The HSV vector can be an HSV-based genome editing vector as described in US Patent Publication No. 2018 / 0148711. The HSV vector can also be an HSV vector that does not express toxic HSV genes in non-complementing cells and includes a genome that includes one or more transgenes as described in US Patent Publication No. 2019 / 0276845.

[0033] The terms "HSV-2 persistent virus" and "HSV-2 mutant" are used interchangeably herein.

[0034] As used herein, the terms "cell membrane fusion" and "fusion" refer to the fusion of the outer membranes of at least two cells, for example, two adjacent cells.

[0035] As used herein, the term "enhanced fusogenic activity" refers to an enhancement, increase, strengthening, argumentation, amplification, or a combination thereof, of cell membrane fusion.

[0036] The term "persistent" as used herein refers to a viral infection characterized as one in which the virus has not been eliminated but remains in certain cells of an infected individual. A persistent infection can involve both asymptomatic and productive infection stages without rapidly killing or even causing damage to the host cells.

[0037] The term "vector" as used herein refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. The inserted nucleic acid sequence is termed "exogenous" either when it is foreign to the cell into which the vector is introduced, or when it is homologous to a sequence in the cell but is in a location within the host cell nucleic acid where die sequences are not normally found. Viral vectors are packaged with viral proteins and are capable of infecting cells. Vectors can be constructed by standard recombinant techniques. In general, these include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989), and references cited therein. Virological considerations are also reviewed in Coen DM, Molecular Genetics of Animal Viruses in Virology, 2nd Edition, BN Fields (editor), Raven Press, NY (1990), and references cited therein.

[0038] The term "expression vector" refers to any type of genetic construct that contains a nucleic acid encoding an RNA that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences." Control sequences refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that serve other functions as well, as described below.

[0039] A "promoter" is a control sequence, a region of a nucleic acid sequence where transcription initiation and transcription rate are controlled. It may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind to initiate or regulate temporal and spatial transcription of the nucleic acid sequence. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to the nucleic acid sequence to control transcription initiation and / or expression of that sequence. Exemplary non-limiting promoters include constitutive promoters, tissue-specific promoters, tumor-specific promoters, or endogenous promoters under the control of exogenous inducible elements.

[0040] The term "constitutive promoter" as used herein refers to a promoter that drives the expression of a gene or polynucleotide in a continuous temporal manner throughout the cell cycle. A constitutive promoter can be cell or tissue type specific, so long as it operates in a continuous temporal manner throughout the cell cycle to drive the expression of a gene or polynucleotide with which it is associated. Exemplary non-limiting constitutive promoters include the immediate early cytomegalovirus (CMV) promoter, the SV40 early promoter, the RSV LTR, the beta chicken actin promoter, and the HSV TK promoter.

[0041] The term "enhancer" refers to a cis-acting regulatory sequence involved in controlling the transcriptional activation of a nucleic acid sequence.

[0042] As used herein, the term "effective" or "therapeutically effective" refers to inhibiting or inhibiting the progression of a symptom, inhibiting, inhibiting, or preventing the onset of a disease, inhibiting, inhibiting, or preventing the spread of a disease, ameliorating at least one symptom of a disease, or a combination thereof.

[0043] The term "patient" or "subject" refers to a mammal, such as a human or a domestic animal (e.g., a dog or cat). In a preferred embodiment, the patient or subject is a human.

[0044] The term "serum" or "serum" refers to the fluid derived from blood that remains when the blood cells and clotting proteins have been removed.

[0045] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" as used herein refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or human, as appropriate. The phrase "pharmacologically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0046] The term "unit dose" refers to physically discrete units suitable for use in subjects, each unit containing a predetermined quantity of a therapeutic composition calculated to produce a desired response in association with its administration (i.e., the appropriate route and treatment regimen).

[0047] The present invention may be utilized as a stand-alone therapy or in conjunction with other therapeutic approaches, including pharmaceutical treatments and surgery.

[0048] Vector construction The HSV vector contains a transgene encoding a desired protein, and in certain embodiments further comprises regulatory sequences such as a promoter. The recombinant virus may further be composed of some or all of the following components:

[0049] vector Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Methods for the construction of engineered viruses and DNA vectors are known in the art. In general, these include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989), and references cited therein. Virological considerations are also found in Coen DM, Molecular Genetics of Animal Viruses in Virology, 2001, pp. 1171-1175, 1997. nd Edition, BN Fields (editor), Raven Press, NY (1990), and the references cited therein.

[0050] Expression vectors can contain a variety of "control sequences." Control sequences refer to nucleic acid sequences necessary for the transcription, and possibly translation, of an operably linked coding sequence (such as a transgene) in a particular host cell. In addition to control sequences that govern transcription and translation, DNA vectors, expression vectors, and viruses can contain nucleic acid sequences that serve other functions as well.

[0051] In one embodiment, the HSV-based vector does not express ICP0, ICP4, ICP22, and ICP27 as immediate early genes. Such vectors can be prepared as described in U.S. Patent Publication No. 2019 / 0276845, which is incorporated herein by reference.

[0052] The HSV vector can be an amplicon packaged into HSV-1 or HSV-2 particles. HSV amplicons are unique gene delivery vectors. They are plasmid-based constructs that contain a copy of the HSV replication origin and a packaging signal in addition to the therapeutic gene(s). When the amplicon construct is co-delivered into a cell with HSV virus as a helper, it is replicated (due to the presence of the replication origin), and then the replicated amplicon sequence is packaged into a virus particle (due to the presence of the packaging signal). The packaged amplicon has the same gene transfer properties as the helper virus. All HSV viruses described in this disclosure can be applied as helper viruses for HSV amplicon packaging for gene delivery.

[0053] 1. Promoters and enhancers Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, although in some promoters that lack a TATA box (e.g., the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late genes), separate elements that cover the start site itself serve to anchor the start position. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0054] Spacing between promoter elements is often flexible, so that promoter function is preserved even if elements are flipped or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with enhancers.

[0055] A promoter may be naturally associated with a nucleic acid sequence, as can be obtained by isolating the 5' non-coding sequences located upstream of a coding segment and / or exon. Similarly, an enhancer may be naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages are obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or from prokaryotic or eukaryotic cells, as well as promoters or enhancers that are "non-naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA constructs include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in conjunction with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as, for example, mitochondria, chloroplasts, etc., may be used as well.

[0056] Naturally, it will be important to employ a promoter and / or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. The promoter employed may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high levels of expression of the introduced DNA segment, such as would be advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0057] Furthermore, any promoter / enhancer combination can be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0058] The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, are well known to those of skill in the art. Non-limiting examples of such regions include the human LIMK2 gene (Nomoto et al. (1999) Gene 236(2):259-271), the somatostatin receptor 2 gene (Kraus et al., (1998) FEBS Lett. 428(3):165-170), the mouse epididymal retinoic acid binding gene (Lareyre et al., (1999) J. Biol. Chem. 274(12):8282-8290), human CD4 (Zhao-Emonet et al., (1998) Biochem. Biophys. Acta, 1442(2-3):109-119), mouse α2(XI) collagen (Tsumaki, et al., (1999) J. Biol. Chem. 274(12):8282-8290), and the human IL-1 (IL-2) (IL-3) (IL-4) (IL-5) (IL-6) (IL-7) (IL-8) (IL-9) (IL-10) (IL-11) (IL-12) (IL-13) (IL-14) (IL-15) (IL-16) (IL-17) (IL-18) (IL-19)). al., (1998), J. Biol. Chem. 273(36):22861-4), D1A dopamine receptor gene (Lee, et al., (1997), DNA Cell Biol. 16(11):1267-1275), insulin-like growth factor II (Vu et al., (1997) Biophys Biochem Res. Comm. 233(1):221-226), and human platelet endothelial cell adhesion molecule 1 (Almendro et al., (1996) J. Immunol. 157(12):5411-5421).

[0059] 2. Initiation signals and internal ribosome binding sites Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be able to readily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and initiation codons may be either natural or synthetic. The efficiency of expression may be enhanced by including appropriate transcriptional enhancer elements.

[0060] In certain embodiments of the present invention, the use of internal ribosome entry site (IRES) elements is used to generate multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at an internal site. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, generating polycistronic messages. The IRES element allows each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819).

[0061] 3. Termination signal The vector or construct of the present invention generally comprises at least one termination signal. A "termination signal" or "terminator" consists of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve a desired message level.

[0062] In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow for site-specific cleavage of the new transcript to expose a polyadenylation site. This transmits a signal to a specialized endogenous polymerase to add a series of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is contemplated that the terminator contains a signal for cleavage of the RNA, and that the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may serve to enhance message levels and minimize read-through from the cassette to other sequences.

[0063] Terminators contemplated for use in the present invention include any known transcription terminator described herein or known to one of skill in the art, including, but not limited to, a genetic termination sequence, such as, for example, the bovine growth hormone terminator, or a viral termination sequence, such as, for example, the SV40 terminator. In certain embodiments, the termination signal can be the absence of a transcribable or translatable sequence, such as by sequence truncation.

[0064] 4. Polyadenylation Signal In expression, particularly in eukaryotic expression, the sequence typically includes a polyadenylation signal that provides for proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be critical to the successful implementation of the present invention, and any such sequence may be used. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, both of which are convenient and known to function well in a variety of target cells. Polyadenylation may improve the stability of the transcript or facilitate cytoplasmic transport.

[0065] 5. Selectable and Screenable Markers In certain embodiments of the present invention, cells containing the nucleic acid constructs of the present invention can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker would confer an identifiable change to the cell that allows easy identification of cells containing the expression vector. Generally, a selection marker is one that confers a property that allows for selection. A positive selection marker is one whose presence allows for its selection, and a negative selection marker is one whose presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0066] Typically, the inclusion of a drug selection marker (e.g., genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol) is a useful selection marker for cloning and identification of transformants. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of a condition, other types of markers are contemplated, including screenable markers such as GFP based on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art will also know how to use immunological markers, possibly in conjunction with fluorescence-activated cell sorting (FACS) analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0067] The vector is introduced into the initially infected cell by a suitable method. Such methods for nucleic acid delivery for transformation of an organelle, cell, tissue, or organism for use in the present invention are intended to include virtually any method by which a nucleic acid (e.g., an HSV vector) can be introduced into an organelle, cell, tissue, or organism, as described herein or known to one of skill in the art. Non-limiting exemplary methods include direct delivery of DNA by ex vivo transfection, injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859), microinjection (U.S. Pat. No. 5,789,215), electroporation (U.S. Pat. No. 5,384,253), calcium phosphate precipitation, DEAE-dextran followed by polyethylene glycol, direct sonication, liposome-mediated transfection, receptor-mediated transfection, biolistics ( Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880), agitation with silicon carbide fibers (U.S. Pat. Nos. 5,302,523 and 5,464,765), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055), PEG-mediated transformation of protoplasts (U.S. Pat. Nos. 4,684,611 and 4,952,500), desiccation / inhibition-mediated DNA uptake, and any combination of these methods or other methods known to those of skill in the art. The composition can also be delivered to mammalian cells by administering it systemically (eg, intravenously) in a pharma- ceutically acceptable excipient.

[0068] Methods for delivering DNA vectors into cells 1. Ex Vivo Transduction Methods for transfecting cells and tissues removed from an organism in an ex vivo setting are known to those skilled in the art. Thus, the present invention contemplates that cells or tissues can be removed and transfected ex vivo using the nucleic acids and compositions described herein. In certain aspects, transplanted cells or tissues can be placed within an organism. In some embodiments, the nucleic acid is expressed in the transplanted cells or tissues.

[0069] 2. Injection In certain embodiments, the nucleic acid may be delivered to an organelle, cell, tissue, or organism via one or more injections (i.e., needle injection), for example, subcutaneously, intradermally, intramuscularly, intravenously, intraperitoneally, etc. Injection methods (e.g., injection of a composition comprising a saline solution) are well known to those of skill in the art. Further embodiments of the invention include the introduction of nucleic acid by direct microinjection. The amount of the composition of the invention used may vary depending on the nature of the cell, tissue, or organism to be affected.

[0070] 3. Electroporation In certain embodiments of the invention, nucleic acids are introduced into organelles, cells, tissues, or organisms via electroporation. Electroporation involves exposing a suspension of cells and DNA to a high-voltage electrical discharge. In some variations of this method, certain cell wall degrading enzymes, such as pectin degrading enzymes, are used to make the target recipient cells more susceptible to transformation by electroporation than untreated cells (US Pat. No. 5,384,253). Alternatively, the recipient cells can be made more susceptible to transformation by mechanical injury.

[0071] 4. Liposome-mediated Transfection In further embodiments of the invention, the compositions (such as vectors) described herein may be encapsulated in lipid complexes, such as liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers. Nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) are also contemplated.

[0072] In certain embodiments of the invention, the liposome may be complexed with Sendai virus (HVJ), which has been shown to promote fusion with cell membranes and facilitate cell entry of liposome-encapsulated DNA (Kaneda et al., (1989) Science 20;243(4889):375-8). In other embodiments, the liposome may be complexed or used in conjunction with nuclear non-histone chromosomal proteins (HMG1) (Kato et al., (1991) J Biol Chem. (1991) February 25;266(6):3361-4). In yet further embodiments, the liposome may be complexed or used in conjunction with both HVJ and HMG1. In other embodiments, the delivery vehicle may include a ligand and a liposome.

[0073] 5. Receptor-mediated Transfection Nucleic acids can be delivered to target cells via receptor-mediated delivery vehicles. This approach utilizes the selective uptake of macromolecules by receptor-mediated endocytosis. Given the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.

[0074] In certain embodiments, the receptor-mediated gene targeting vehicle comprises a receptor-specific ligand and a nucleic acid binding agent. Other embodiments comprise a receptor-specific ligand to which the nucleic acid to be delivered is operably linked. Several ligands have been used in receptor-mediated gene transfer, including epidermal growth factor (EGF), which has been used to deliver genes to squamous cell carcinoma cells, as described in European Patent No. EPO0273085.

[0075] In other embodiments, the nucleic acid delivery vehicle component of the cell-specific nucleic acid targeting vehicle may include a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered are housed within the liposome, and the specific binding ligand is functionally integrated into the liposome membrane. Thus, the liposome specifically binds to the receptor(s) of the target cell and delivers its contents to the cell.

[0076] In yet another embodiment, the nucleic acid delivery vehicle component of the targeted delivery vehicle may be a liposome itself, which preferably comprises one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosylceramide, a galactose-terminal asialganglioside, has been incorporated into liposomes, and increased uptake of insulin gene by hepatocytes has been observed (Nicolau et al., (1987) Methods Enzymol. 149:157-76). It is contemplated that the tissue-specific transformation construct of the present invention can be specifically delivered to target cells in a similar manner.

[0077] 6. Microparticle gun Biolistic techniques can be used to introduce nucleic acids into at least one organelle, cell, tissue, or organism (U.S. Pat. Nos. 5,550,318, 5,538,880, 5,610,042, and PCT Application WO94 / 09699). This method relies on the ability to accelerate microprojectiles coated with or containing DNA to high velocities, allowing them to penetrate cell membranes and enter cells without killing them. Microprojectiles can be composed of any biologically inert material, such as tungsten, platinum, or gold. For bombardment, cells in suspension are concentrated onto a filter or solid medium. Alternatively, immature embryos or other target cells may be placed on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the biolistic device on a stopping plate. A wide variety of biolistic techniques useful for implementation according to the present invention are known to those skilled in the art.

[0078] host cell As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms include progeny, which are any subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, a "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector and / or expressing a heterologous gene encoded by the vector. A host cell can be and has been used as a recipient of a vector. A host cell can be "transfected" or "transformed," which refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" or host cell are intended to refer to a cell into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Thus, a recombinant cell is distinguishable from a naturally occurring cell that does not contain a recombinantly introduced nucleic acid.

[0079] A tissue may contain one or more host cells transformed with a fusogenic HSV-2 mutant. A tissue may be part of an organism or may be isolated from an organism. In certain embodiments, tissues include, but are not limited to, adipocytes, alveoli, ameloblasts, nerves, basal cells, blood (e.g., lymphocytes), blood vessels, bone, bone marrow, glial cells, breast, cartilage, cervix, colon, cornea, embryo, endometrium, endothelium, epithelium, esophagus, fascia, fibroblasts, follicles, ganglion cells, glial cells, goblet cells, kidney, liver, lung, lymph nodes, muscles, neurons, ovaries, pancreas, peripheral blood, prostate, skin, small intestine, spleen, stem cells, stomach, and testes.

[0080] In certain embodiments, the host cell or tissue may be comprised in at least one organism, which may be, but is not limited to, a prokaryote (e.g., fungus, archaea) or a eukaryote, as will be appreciated by those of skill in the art.

[0081] Numerous cell lines and cultures are available for use as host cells and are commercially available through organizations such as the American Type Culture Collection (ATCC). The appropriate host can be determined by one of skill in the art based on the vector backbone and the desired outcome. Exemplary non-limiting cell types available for vector replication and / or expression include bacteria such as E. coli (e.g., E. coli strains RR1, LE392, B, X1776 (ATCC No. 31537), W3110, F, lambda, DH5α, JM109, and KC8); bacilli (e.g., Bacillus subtilis); other Enterobacteriaceae (e.g., Salmonella typhimurium, Serratia marcescens), as well as several bacterial hosts and competent cells that are commercially available (e.g., SURE® competent cells and SOLOPACK™ Gold cells (STRATAGENE®, La Jolla, Calif.)). Non-limiting examples of eukaryotic host cells for replication and / or expression of vectors include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12.

[0082] Some vectors may employ control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art will further understand the conditions under which all of the above host cells may be incubated to maintain them and allow replication of the vectors. Techniques and conditions that allow large-scale production of vectors, and production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides, are also understood and known.

[0083] Packaging and propagation of viral vectors 1. Viral Packaging In certain embodiments of the present invention, transgenes can be inserted into viruses via homologous recombination. Typically, this is done by co-transfecting plasmid DNA containing transgenes with purified HSV (e.g., HSV-2) genomic DNA into Vero cells using Lipofectamine. Recombinant viruses are then identified (typically by screening viral plaques for the presence of a selection marker) and plaques containing the transgene are selected. Selected recombinant viruses are then characterized in vitro to confirm that the transgene is correctly inserted into the HSV genome.

[0084] 2. Viral Stock Preparation Once a recombinant HSV (e.g., HSV-2) mutant virus is selected, virus stocks can be prepared as follows: Vero cells are grown in 10% fetal bovine serum (FBS) and infected with 0.01 plaque-forming units (pfu) per cell. Virus is then harvested from the cells after 2 days by repeated freezing and thawing and sonication. The harvested virus is then purified as described (Nakamori, et al., (2003) Clinical Cancer Res. 9(7):2727-2733). The purified virus is then titrated, aliquoted, and stored at -80°C until use.

[0085] Protein Expression Systems Protein expression systems can be utilized in the production of the DNA vector compositions of the present invention, for example, to express the polypeptides encoded by the transgenes for functional studies. Numerous expression systems exist that contain at least some or all of the compositions described above. Prokaryotic and / or eukaryotic based systems can be used to produce nucleic acid sequences, or their cognate polypeptides, proteins, and peptides, for use with the present invention. Many such systems are commercially and widely available.

[0086] Insect cell / baculovirus systems are capable of producing high levels of protein expression of heterologous nucleic acid segments, such as those described in U.S. Pat. Nos. 5,871,986 and 4,879,236, and are commercially available (e.g., CLONTECH, Inc. Mountain View, Calif.).

[0087] Other examples of commercially available expression systems include a synthetic ecdysone-inducible receptor, or pET expression system, or E. coli expression system (STRATAGENE, LaJolla, Calif.); a tetracycline-regulated expression system, an inducible mammalian expression system using the full-length CMV promoter, or a yeast expression system designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica (INVITROGEN, Carlsbad, Calif.).

[0088] It is contemplated that the protein, polypeptide, or peptide produced by the method of the invention may be "overexpressed", i.e., expressed at an increased level compared to its natural expression in the cell. Such overexpression may be assessed by a variety of methods, including radiolabeling and / or protein purification. However, simple and direct methods are preferred, such as those involving SDS / PAGE and protein staining or Western blotting, followed by quantitative analysis such as densitometric scanning of the resulting gel or blot. A specific increase in the level of a recombinant protein, polypeptide, or peptide compared to the level in a native cell indicates overexpression, as does the relative abundance of the particular protein, polypeptide, or peptide compared to other proteins produced by the host cell (e.g., as seen on a gel).

[0089] Pharmaceutical Compositions and Routes of Administration The compositions of the present invention can be administered as pharmaceutical compositions comprising modified HSV-based vectors containing one or more copies of therapeutic genes as described herein. The compositions of the present invention include classical pharmaceutical formulations. In general, the compositions of the present invention can be administered as pharmaceuticals by dissolving or dispersing the compositions in a pharma-ceutically acceptable carrier or aqueous medium. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compositions of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-disease agents, may also be incorporated into the pharmaceutical composition. Administration of the compositions will be via any common route, so long as the target cells are available via that route. Exemplary routes of administration include oral, nasal, buccal, rectal, vaginal, or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or direct intratumoral injection. The pharmaceutical formulations, dosages, and routes of administration of the compositions of the present invention are described herein. Administration can be by systemic administration or by intracerebroventricular or intraperitoneal injection.

[0090] Pharmaceutical formulations of HSV vectors The composition or HSV vector of the present invention can be prepared as a pharmacologically acceptable formulation. Typically, the composition or vector is mixed with an excipient that is pharma- ceutically acceptable and compatible with the virus. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if necessary, the preparation may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffers, adjuvants, or immuno-enhancing agents, which enhance the effectiveness of the virus mutant (see Remington's Pharmaceutical Sciences, Gennaro, AR et al., eds., Mack Publishing Co., pub., 18th ed., 1990). For example, a typical pharma-ceutically acceptable carrier for injection purposes may contain 50 mg to about 100 mg of human serum albumin per milliliter of phosphate-buffered saline. Additional non-limiting exemplary non-aqueous solvents suitable for use in the formulation of pharmacologically acceptable compositions include propylene glycol, polyethylene glycol, vegetable oil, sesame oil, peanut oil, and injectable organic esters such as ethyl oleate.Exemplary non-limiting aqueous carriers include water, aqueous solutions, saline, parenteral vehicles (e.g., sodium chloride, Ringer's dextrose).Intravenous vehicles include fluid and nutrient replenishers.Determining the pH and exact concentration of various components of pharmaceutical compositions is routine and within the knowledge of those skilled in the art (see Goodman and Gilman's The Pharmacological Basis for Therapeutics, Gilman, AGet al., eds., Pergamon Press, pub., 8th ed., 1990).

[0091] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other sterile ingredients as required and as described above. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients as described above.

[0092] In certain embodiments, when the present invention is used to treat neurological conditions, convection-enhanced delivery (CED) can be utilized. Convection-enhanced delivery (CED) involves using fine catheters and precisely controlled infusion rates to distribute therapeutic agents directly into the brain extracellular space by bulk flow, presumably along the same extracellular pathways along which glioma cells may migrate. In contrast to techniques of drug delivery that rely on diffusion to achieve adequate drug distribution, such as carmustine-impregnated biodegradable polymers, it is possible to use CED to distribute drugs uniformly across the potentially large volume of the brain, regardless of the molecular size of the therapeutic agent. It is therefore an ideal technique for the administration of viral vector-mediated gene therapy to the brain of a patient.

[0093] In some embodiments, the HSV-based gene delivery vector may be a composition further comprising cerebrospinal fluid (CSF), in particular artificial cerebrospinal fluid. Artificial cerebrospinal fluid is well known in the art and is a fluid that mimics natural CSF, in particular with respect to its salt content. Preferably, the composition comprises NaCl at a concentration similar to that found in natural CSF, i.e., the concentration is preferably within 15%, more preferably within 10%, of the concentration in natural CSF. Preferably, the composition comprises NaHCO3 at a concentration similar to that found in natural CSF, i.e., the concentration is preferably within 15%, more preferably within 10%, of the concentration in natural CSF. Preferably, the composition comprises KCl at a concentration similar to that found in natural CSF, i.e., the concentration is preferably within 15%, more preferably within 10%, of the concentration in natural CSF. Preferably, the composition contains NaH2PO4 at a concentration similar to that found in natural CSF, i.e., the concentration is preferably within 15%, more preferably within 10% of the concentration in natural CSF. Preferably, the composition contains MgCl2 at a concentration similar to that found in natural CSF, i.e., the concentration is preferably within 15%, more preferably within 10% of the concentration in natural CSF. Preferably, the composition contains glucose at a concentration similar to that found in natural CSF, i.e., the concentration is preferably within 15%, more preferably within 10% of the concentration in natural CSF. Alternatively, the artificial CSF may omit glucose to reduce the possibility of bacterial growth in any catheter used to administer the composition to a subject.

[0094] The composition may further comprise other active agents. The pharmaceutical composition of the present invention may also comprise any pharma- ceutically acceptable carrier, adjuvant, or vehicle. The pharma-ceutically acceptable carrier, adjuvant, and vehicle that can be used in the pharmaceutical composition include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates, glycine, sorbic acid, potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and polyethylene glycol.

[0095] The pharmaceutical compositions of the present invention may be administered by any suitable route, but are preferably administered by injection, particularly via a neural catheter, and especially by convection-enhanced delivery. The pharmaceutical compositions may contain any conventional non-toxic pharma- ceutical acceptable carriers, adjuvants, or vehicles.

[0096] Route and dose of HSV vector administration The mutant virus composition may be delivered by any route that provides access to the target tissue. Exemplary non-limiting routes of administration may include oral, nasal, buccal, rectal, vaginal, topical, or by injection (including orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or intracerebroventricular injection). Typically, the virus mutant will be prepared as an injectable, either a liquid solution or suspension. Solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified.

[0097] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known techniques using suitable dispersing or wetting agents (such as, for example, Tween® 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives, particularly in their polyoxyethylated form, are useful in the preparation of injectables, as are natural pharma-ceutically acceptable oils such as olive oil or castor oil. These oil solutions or suspensions may also contain a diluent or dispersant such as a long-chain alcohol such as Ph. Helv or a similar alcohol.

[0098] For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0099] Those skilled in the art will recognize that the best treatment regimen for providing therapy using the compositions of the present invention can be determined straightforwardly. This is not a matter of experimentation, but rather an optimization problem that is routinely performed in the medical field. For example, in vivo studies in mice provide a starting point to begin optimizing the dosage and delivery regime. The frequency of injections may initially be once a week. However, this frequency may be optimally adjusted from 1 day to every 2 weeks to monthly, depending on the results obtained from initial clinical trials and the needs of the particular patient. Human dosages may initially be determined by extrapolating from the amount of composition used in mice.

[0100] Dosage The amount of viral vector delivered will depend on several factors, including the number of treatments, the subject being treated, the ability of the subject's immune system to synthesize anti-viral antibodies, the target tissue to be destroyed, and the degree of protection desired. The precise amount of viral composition to be administered depends on the judgment of the practitioner and is unique to each individual. However, suitable dosages are within the range of 10 5 Plaque forming units (pfu) to 10 10 In certain embodiments, the viral DNA dosage is in the range of about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , up to 10 10 It may be pfu.

[0101] Non-viral DNA vector formulations In addition to the above-mentioned formulation for viral pharmaceutical preparation, non-viral DNA vector can also be prepared as sterile powder for preparing pharmacologically acceptable sterile solution.The typical method for preparing sterile powder includes vacuum drying and freeze-drying technology, which allows the powder of active ingredient and any additional desired ingredients to be obtained from its solution that has been previously sterile-filtered.

[0102] Routes and Dosages for Administration of Non-Viral DNA Vectors Several methods are contemplated for the delivery of non-viral vectors to introduce the polynucleotide of the present invention into mammalian cells. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high-velocity microprojectiles, and receptor-mediated transfection as described above. Some of these techniques can be successfully adapted for in vivo or ex vivo use.

[0103] In some embodiments of the invention, the expression vector may simply consist of naked recombinant DNA or a plasmid containing the polynucleotide. Introduction of the construct may be by any of the methods described herein for physically or chemically permeabilizing the cell membrane. This applies particularly to in vitro transfer, but may also be applied for in vivo use.

[0104] In other embodiments, the delivery vehicle may include a ligand and a liposome. For example, Nicolau et al. observed increased uptake of the insulin gene by hepatocytes using lactosylceramide, a galactose-terminated asialganglioside, incorporated into liposomes (Nicolau et al., (1987) Methods Enzymol. 149:157-76). It is therefore possible that nucleic acids encoding specific genes can also be specifically delivered to a cell type by any number of receptor-ligand systems, with or without liposomes. For example, epidermal growth factor (EGF) can be used as a receptor for mediated delivery of nucleic acids to cells that show EGF receptor upregulation (as described in European Patent No. EP0273085), and mannose can be used to target the mannose receptor on hepatocytes.

[0105] In certain embodiments, DNA introduction can be more easily performed under ex vivo conditions.Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of nucleic acid to the cells in vitro, and then the return of the modified cells to the animal.This can include surgical removal of tissue / organs from the animal, or primary culture of cells and tissues.

[0106] Dosage In certain embodiments, the dosage is about 10 3 pfu / kg body weight ~ approx. 10 8 It is contemplated that the dosage may vary between about 10 pfu / kg body weight. In certain embodiments, the dosage is about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , up to 10 8 pfu / kg body weight. Of course, this dosage may be adjusted upwards or downwards, as is routinely done in such treatment protocols, depending on the results of initial clinical trials and the needs of a particular patient.

[0107] Treatment method Yet another embodiment is a method of treating a disease in a patient in need thereof, comprising administering to the patient a composition described herein or a non-replicating HSV-based vector described herein. The disease or disorder may be a neurological disease or disorder. In one embodiment, the disease or disorder is not a neoplastic disease or disorder (e.g., the disease or disorder is not cancer). The composition or HSV-based vector may be administered systemically, intramuscularly, intradermally, subcutaneously, or parenterally, or by intracerebroventricular or intraperitoneal injection. In one embodiment, the patient has been exposed to, or has been vaccinated against, or has HSV-1 and / or HSV-2.

[0108] Yet another embodiment is a method of vaccinating a patient against a malignant or infectious disease comprising administering to the patient a composition described herein or a non-replicating HSV-based vector described herein, in one embodiment, the transgene is one useful for vaccinating a patient.

[0109] Combination treatment Other gene therapies can also be used in conjunction with the compositions and methods described herein as a combination therapy for the treatment of a disease or disorder. Gene therapy as a combination therapy relies on the delivery and expression of a therapeutic gene separate from the HSV vector described herein. The gene therapy can be administered before, after, or simultaneously with the HSV vector described herein. Exemplary non-limiting targets for gene therapy include immunomodulatory cells, agents that affect the upregulation of surface receptors and GAP junctions, cytostatic and differentiating agents, or inhibitors of cell adhesion. Exemplary non-limiting immunomodulatory genes that can be used as part of a gene therapy in combination with the present invention include tumor necrosis factor, interferon alpha, beta, and gamma, IL-2 and other cytokines, F42K and other cytokine analogs, or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines.

[0110] Hormonal therapy may also be used in conjunction with the present invention. The use of hormones may be used to treat a variety of disorders in order to lower the levels or block the effects of certain hormones, such as testosterone or estrogen. EXAMPLES

[0111] The following examples are included to demonstrate aspects of the invention. Although the examples are presented with respect to oncolytic viruses, one of skill in the art will understand that the beneficial effects of passaging and inclusion of the extracellular CD47 domain also apply to any of the HSV-based gene delivery vectors described herein.

[0112] It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and are therefore believed to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0113] Example 1: Construction of FusOn-H2 FusOn-H2 was constructed as described in Example 1 of US Pat. No. 10,039,796. To systematically mutate most of the neutralizing epitopes in both gB and gD, as well as to enhance the complement antagonism ability of gC, FusOn-H2 was subjected to a series of selections in the presence of anti-HSV-2 serum. FusOn-H2 was first subjected to selection in cell culture in the presence of a mixture of sera collected from five rats that had been vaccinated with HSV and had high anti-HSV antibodies in their blood. One of the main reasons for starting this selection with rat sera is that they contain both natural immunoglobulins and mannan-binding lectin (MBL), which can activate complement against HSV, whereas mouse and human sera contain only one of these two activation mechanisms (MBL for mouse and natural immunoglobulin for human, respectively) (Wakimoto et al., 2002). After seven successive rounds of selection under this rat serum mixture, one human serum containing high levels of anti-HSV-2 antibodies was added to the rat serum and selection continued.

[0114] During the selection process, virus infectivity in the presence of anti-HSV serum was monitored periodically, and the results showed a continuous improvement over unselected FusOn-H2 in terms of resistance to neutralizing antibodies. Figure 1 shows one of the test results, which was performed after the virus had undergone a total of nine rounds of selection (i.e., seven rounds of selection with rat serum and two rounds of selection with a mixture of rat and human serum). The results showed that the trained virus (designated FusOn-SS9) was 28.5-fold and 27.2-fold more resistant to the neutralizing effect of human and rat anti-HSV-2 serum, respectively.

[0115] To enable FusOn-H2 with the ability to evade host clearance by the mononuclear phagocyte system (MPS) in the blood during systemic delivery, we explored the possibility of genetically grafting CD47, a "don't eat me" signal molecule, onto the viral membrane envelope. Macrophages are the main phagocytes involved in the rapid clearance of HSV particles, and it has been reported that macrophage depletion can significantly improve the therapeutic efficacy of oncolytic HSV (Fulci et al., 2007). The phagocytic activity of macrophages is controlled by both positive and negative regulatory mechanisms. The interaction between CD47 and its receptor, SIRPα, provides a strong negative regulatory signal ("don't eat me signal") to macrophages (Kinchen and Ravichandran, 2008).

[0116] HSV encodes several glycoproteins that assemble on the surface of the viral envelope. They include glycoprotein C (gC), gB, gD, gH, and gL. Each of them could serve as a candidate molecule for incorporating the extracellular domain (ECD) of mouse CD47 (mCD47) so that it could be grafted onto the surface of the viral envelope. Glycoprotein C (gC) was chosen here because, unlike the other glycoproteins mentioned, it is not essential for viral infection. Therefore, modifying it to incorporate mCD47 would not risk altering the natural tropism of the oncolytic virus. The ECD (shown in the table below) of mCD47 (aa19-141) (SEQ ID NO: 1) was first inserted at the N-terminus of gC to generate a chimeric form of gC (cgC), whose expression was driven by the CMV IE promoter. The sequences of full-length mouse CD47 (SEQ ID NO: 2) and human CD47 (SEQ ID NO: 4) are provided in the table below. [Table 1-1] [Table 1-2] [Table 1-3]

[0117] To allow convenient detection of the chimeric molecule, an HA (hemagglutinin) tag was included in cgC. For the purpose of easy identification of recombinant viruses and for in vivo imaging purposes, cgC was linked to another gene cassette containing the EGFP-luciferase gene. These two gene cassettes were inserted together into the backbone of FusOn-H3, which was derived from an HSV-2 based oncolytic virus (FusOn-H2) by deleting the GFP gene from the virus. FusOn-H2 was constructed by deleting the N-terminal region of the ICP10 gene and replacing it with GFP, giving it the ability to selectively replicate in and kill tumor cells. Recombinant viruses were identified by selecting GFP-positive plaques. Each individually selected virus was concentrated to homogeneous GFP positivity by multiple rounds of plaque purification. The newly generated virus was named FusOn-CD47-Luc (Figure 2A). We also constructed control viruses in which only the EGFP-Luc gene cassette was inserted into the backbone of FusOn-H3, FusOn-Luc (Figure 2B). All selected viruses maintain the fusion properties of the parent FusOn-H2.

[0118] An interesting recent study found that coating of nanoparticles with CD47 mimetic peptides could help to avoid phagocytic clearance by MPS (Rodriguez et al., 2013). We explored the possibility of genetically grafting the extracellular domain of the CD47 molecule onto the membrane envelope of FusOn-H2 to enable it to escape from MPS for systemic delivery. Figure 2 shows the virus construction strategy. In vitro assays show that coating the viral particles with the CD47 extracellular domain via recombinant gC makes the virus more resistant to phagocytosis (Figure 3). When tested in vivo in a mouse colon cancer model, the results showed that FusOn-CD47 was delivered to the tumor site by the systemic route 15 times more effectively than FusOn-Luc. Furthermore, the data also showed that FusOn-CD47 could persist significantly longer than FusOn-Luc at the tumor site after systemic delivery.

[0119] First, the expression of the transgene was examined by either flow cytometry or Western blot analysis. For flow cytometry analysis, 293 cells were infected with either FusOn-CD47-Luc or FusOn-Luc at 1 pfu / cell. After 24 hours, the cells were labeled with either PE-conjugated anti-mCD47 antibody or rabbit anti-HA tag antibody. Goat anti-rabbit antibody conjugated with FITC was added for detection of the HA tag. Both CD47 and HA tag labeled cells were subjected to flow cytometry analysis. The results in Figure 2A show that both anti-mCD47 antibody and anti-HA tag antibody could easily detect cells infected with FusOn-CD47-Luc, but could not detect cells infected with FusOn-Luc. For Western blot analysis, 293 cells were infected with these two viruses in the same way, or transfected with a plasmid expressing cgC or a plasmid expressing wild-type gC without the HA tag. After 24 hours, cell lysates were prepared and subjected to Western blot analysis. The results in Figure 2B show that cgC is abundantly expressed by FusOn-CD47-Luc, but not by FusOn-Luc.

[0120] We then compared the quantification of Luc gene expression from these two viruses. Vero, CT26, and 4T1 cells were infected with either FusOn-CD47-Luc or FusOn-Luc at 1 pfu / cell. After 24 hours, cells were harvested for measurement of luciferase activity. The results in Figure 3A showed that luciferase activity from cells infected with these two viruses was at approximately the same level. Another experiment was then performed to determine whether implanting oncolytic HSV with the ECD of mCD47 in an in vitro setting would allow the virus to be phagocytosed by phagocytes and avoid clearance. Mouse splenocytes were used as a source of fresh phagocytes because the spleen is the largest unit of the mononuclear phagocyte system. Vero cells were infected with FusOn-CD47-Luc or FusOn-Luc in the presence or absence of mouse splenocytes. After 48 hours, cells were harvested for quantitative measurement of virus yield by plaque assay. The results in Figure 3B showed that in wells without splenocytes, both FusOn-CD47-Luc and FusOn-Luc replicated well, and the virus yields in these two wells were similar. However, in wells with splenocytes, the yield of FusOn-Luc was significantly reduced, while the replication of FusOn-CD47-Luc was only slightly affected. These results indicate that the presence of mCD47 ECD on the virus particles endows the virus with the ability to resist the influence of macrophages and possibly some other immune cells during viral infection.

[0121] To determine the ability of the incorporated mCD47 ECD to enable systemic delivery of the virus, CD26 murine colon cancer cells were implanted into the right flank of immune-competent Balb / c mice. When the tumors reached a size of about 8 mm in diameter, we systemically injected each mouse with 2×106 pfu of either FusOn-CD47-Luc or FusOn-Luc. The mice were monitored for luciferase activity by an IVIS Spectrum system. Monitoring began on day 2 and was then performed daily until the signal completely disappeared. The results in FIG. 4 show that on day 2 after virus administration, the imaging signal from FusOn-CD47-Luc was about 1.5 logs stronger than that from FusOn-Luc. This indicates that the former was delivered to the tumor site more efficiently by the systemic route than the latter. Furthermore, FusOn-CD47-Luc appears to remain in the tumor tissue substantially longer than FusOn-Luc. By day 5, imaging signals were barely detectable in tumors from mice that received FusOn-Luc, whereas signals in tumors from FusOn-CD47-Luc were detectable until day 7. Nevertheless, because this HSV-2-based oncolytic virus does not grow well in CT26 tumor cells, neither virus showed significant amplification in tumor tissue. Interestingly, when several mice were imaged on day 1 after virus delivery, prominent imaging signals were transiently detected in the liver. These signals completely disappeared by day 2.

[0122] To determine whether FusOn-CD47-Luc is superior to FusOn-Luc for systemic delivery in other tumor models, the above experiment was repeated but in mice bearing tumors in the right flank established from implantation of 4T1 mouse breast tumor cells, in which FusOn-H2 was found to be able to grow moderately. Furthermore, 4T1 tumor cells secrete macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF), which can enhance macrophage infiltration and phagocytosis. This allows the CD47-mediated evasion strategy to be tested more vigorously. The IVIS imaging results in Figure 5A indeed showed that the signal in 4T1 tumors was generally lower than that detected in CT26 tumors (shown in Figure 4). Nevertheless, the results again showed that FusOn-CD47-Luc could be delivered to local tumors more efficiently than FusOn-Luc by the systemic route. By day 2, the difference in image signal intensity between these two viruses was about 1.5 logs. The largest difference was recorded on day 4, when the image signal from FusOn-Luc had decreased to near background levels and FusOn-CD47-Luc had reached its highest level. These data again demonstrated that CD47 modification allowed the virus to be delivered more efficiently by the systemic route and, once there, the virus persisted longer in the tumor tissue than the control virus.

[0123] To generate a product of FusOn-H2 that can withstand all three major restriction factors in the bloodstream: neutralizing antibodies, complement, and phagocytes, we subjected FusOn-CD47 to anti-HSV-2 serum selection as described above. As described above, it was subjected to seven serial sections with anti-HSV rat serum, and then 23 serial passages in the presence of one human serum with extremely high levels of anti-HSV-2 antibodies in addition to the rat serum. Again, several tests were performed during selection to determine whether the virus had acquired the ability to resist the neutralizing effect of immune serum. Figure 5 shows one of the results from such a test, which first subjected FusOn-CD47 to seven sections with rat serum, followed by 17 sections with human serum in addition to the rat serum. The results clearly show that, similar to FusOn-SS9, the trained FusOn-CD47 (designated FusOn-CD47-SS24) has acquired a substantial ability to maintain infectivity in the presence of immune anti-HSV-2 serum.

[0124] To ensure a more severe resistance to neutralizing antibodies in humans, we subjected the viruses to three more sections using a mixture of HSV-2 positive sera from 12 individuals (mixed four at a time). The number of selections was eight for batch 1, six for batch 2, and four for batch 3. After these extensive selections, the resulting viruses were plaque purified and six plaque picks were grown for further analysis. These were named HR49-N2-5, HR49-N7, and HR49-N8. First, we tested their ability to resist neutralization from a mixture of the eight human sera used for selection. This is the most stringent neutralization test we have performed. For comparison, both FusOn-SS9 and FusOn-CD47-SS24 were included in this experiment. The anti-HSV serum mixture was used at different dilutions to incubate with the indicated viruses, after which the virus infectivity was determined. The results in Figure 6 show that under this stringent neutralization condition (containing a mixture of antiviral sera from multiple individuals), the selected viruses were still able to produce a significant number of plaques, while the unselected viruses did not produce plaques until the serum mixture was diluted 80-fold. The data also showed that subsequent three additional rounds of selection in a human serum mixture further enhanced the ability of the selected viruses to resist the neutralizing effect. The data also showed that some of the selected viruses had a better ability than other viruses to resist neutralization by the mixed human antiviral sera.

[0125] Example 2 The ability of oncolytic virus FusOn-H2 to evade neutralization by anti-HSV antibodies was evaluated with and without insertion of the extracellular domain of CD47 into the N-terminus of gC. In this in vitro experiment, 500 plaque-forming units (pfu) of either FusOn-CD47 or FusOn-Luc were mixed in the presence or absence of diluted (1:40 or 1:160 dilutions) anti-HSV-2 serum, incubated at 37°C, and then applied to Vero cell monolayers to assay infectivity and quantify plaque formation. The results in Figure 7 showed that at a 1:40 dilution, the serum was able to almost completely neutralize the viral infectivity of FusOn-Luc. Even at a 1:160 dilution, the serum was still able to significantly reduce the infection (and therefore the number of plaques formed) of FusOn-Luc. In contrast, FusOn-CD47 was much less neutralized in the presence of the same diluted anti-HSV serum. In the presence of a 1:40 dilution of serum, FusOn-CD47 produced a significant number of plaques (approximately 15% of wells to which anti-HSV serum was not added), whereas FusOn-Luc showed almost no viral plaques.

[0126] In vivo delivery by systemic route was evaluated for FusOn-CD47, FusOn-Luc, and FusOn-SD in Balb / c mice vaccinated with HSV-2 and implanted with CT26 mouse colon cancer cells in the right flank. 7Either FusOn-CD47, FusOn-Luc, or FusOn-SD was delivered systemically to different groups of mice by injection via the tail vein. The mice were imaged on the days indicated in Figure 8 using an IVIS imager. The results showed that FusOn-Luc could not be delivered to the tumor by systemic route in the presence of anti-HSV immunity. In contrast, FusOn-CD47 was readily detected in tumor tissues in the presence of anti-HSV immunity by the same delivery route. Furthermore, FusOn-CD47 persisted in tumor tissues for nearly 8 days after reaching the tumor site, despite the presence of anti-viral immunity. Unexpectedly, CD47 helped the oncolytic virus evade the neutralizing effect by anti-HSV antibodies, contributing to the overall ability of FusOn-SD to evade neutralizing antibodies for systemic delivery. Thus, FusOn-SD performed the best in systemic delivery to the tumor site. Thus, FusOn-SD is useful for patients with HSV-1 and / or HSV-2 antibodies, such as patients who have been vaccinated against HSV-1 and / or HSV-2 or who have HSV-1 and / or HSV-2.

[0127] Surprisingly, gE was found to be absent from FusOn-SD viral particles, as shown in Figures 9A-C.

[0128] Figure 9A is a bar graph showing the absorbance of FusOn-H2, FusOn-SD, and PBS (negative control) after treatment with mouse anti-HSV-2 gE (1:1000 dilution) and HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). More specifically, in Figure 9A, 1 x 10 6pfu of virus (parental FusOn-H2 and FusOn-SD) was coated onto 96-well plates for ELISA assay. Wells coated with PBS served as negative controls. The primary antibody used was mouse anti-HSV-2 gE (1:1000 dilution) and the secondary antibody was HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). Significant readouts were detected with FusOn-H2 but not with FusOn-SD, indicating that gE was present in FusOn-H2 viral particles but not in FusOn-SD.

[0129] Figure 9B is a Western blot showing detection of gE for FusOn-SD and FusOn-H2. 6 pfu of virus (FusOn-H2 and FusOn-SD) were subtracted and run on an acrylamide gel for Western blot analysis. The same antibodies, mouse anti-HSV-2 gE and HPR-conjugated rabbit anti-mouse IgG, used for Figure 9A were used for the Western blot. The results showed a clear band of gE in FusOn-H2, but it was absent in the lane loaded with protein from FusOn-SD. This result supports the findings in Figure 9A.

[0130] Figure 9C is a schematic diagram of the mechanism of NK cell recognition of HSV or HSV-infected cells via gE. The figure shows that gE binds to IgG (either virus-specific or non-virus-specific). CD16a, one of the most important NK cell activation receptors, then binds to the Fc region of IgG, resulting in the activation of NK cells and the clearance of either virus particles or virus-infected cells. This leads to a reduction in the delivery and replication of oncolytic viruses, thus minimizing the therapeutic effect of virotherapy. Because FusOn-SD is not recognized by this mechanism, NK cells do not clear them, and the therapeutic efficacy of FusOn-SD persists.

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[0156] All publications, patents, and patent applications cited herein are incorporated by reference as if set forth herein in their entirety. Although the present invention has been described with reference to exemplary embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of the exemplary embodiments, as well as other embodiments of the present invention, will be apparent to those skilled in the art upon reference to the description. Accordingly, it is intended that the appended claims encompass such modifications and extensions.

[0157] In addition to the various embodiments described herein, the present disclosure includes the following embodiments, numbered E1 through E39: This list of embodiments is presented as an exemplary list, and the application is not limited to these embodiments.

[0158] E1. A non-replicating herpes simplex virus (HSV)-based gene delivery vector, wherein (i) the HSV vector contains a transgene, and (ii) the HSV vector has a membrane envelope comprising glycoproteins, at least one of which contains an extracellular CD47 domain inserted at the N-terminus of the glycoprotein.

[0159] E2. The HSV-based vector of embodiment E1, wherein the HSV vector is an HSV-1 or HSV-2 vector.

[0160] E3. The HSV-based vector of embodiment E1 or E2, wherein the HSV-based vector is an HSV amplicon packaged into a viral particle.

[0161] E4. The HSV-based vector of any one of the preceding embodiments, wherein the glycoprotein is selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L.

[0162] E5. The HSV-based vector of embodiment E4, wherein the glycoprotein is glycoprotein C.

[0163] E6. The HSV-based vector of any one of the preceding embodiments, wherein the transgene is a therapeutic gene.

[0164] E7. The HSV-based vector of any one of the preceding embodiments, wherein said extracellular CD47 domain is an extracellular human CD47 domain.

[0165] E8. A composition comprising an HSV-based vector of any one of the preceding embodiments, wherein the HSV vector is prepared by passaging the HSV vector at least twice with immune serum having elevated levels of anti-HSV antibodies.

[0166] E9. The composition of embodiment E8, wherein said passaging in said immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of said HSV-based vector.

[0167] E10. The composition of embodiment E8 or E9, wherein the extracellular CD47 domain comprises amino acids 19-141 of mouse CD47.

[0168] E11. The composition of embodiment E8 or E9, wherein said extracellular CD47 domain is an extracellular human CD47 domain.

[0169] E12. The composition of any one of embodiments E8-E11, wherein the HSV-based vector is free or substantially free of gE.

[0170] E13. The composition of any one of embodiments E8-E12, wherein the immune serum is a mixture comprising rat serum and human serum with elevated levels of anti-HSV antibodies.

[0171] E14. The composition of any one of embodiments E8-E13, wherein the HSV-based vector is prepared by passaging the HSV vector at least twice in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by at least twice in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

[0172] E15. A composition comprising a non-replicating herpes simplex virus (HSV)-based gene delivery vector, (i) the HSV-based vector comprises a transgene, and (ii) the HSV-based vector is prepared by passaging the HSV-based vector at least twice with immune serum having elevated levels of anti-HSV antibodies.

[0173] E16. The composition of embodiment E15, wherein the HSV-based vector is an HSV-1 or HSV-2 vector.

[0174] E17. The composition of embodiment E15 or E16, wherein said passaging in said immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of said HSV-based vector.

[0175] E18. The composition of any one of embodiments E15-E17, wherein said HSV-based vector is free or substantially free of gE.

[0176] E19. The composition of any one of embodiments E15-E18, wherein the immune serum is a mixture comprising rat serum and human serum with elevated levels of anti-HSV antibodies.

[0177] E20. The composition of any one of embodiments E15-E19, wherein the HSV-based vector is prepared by passaging the HSV-based vector at least twice in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by at least twice in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

[0178] E21. The composition of any one of embodiments E15-E20, wherein the transgene is a therapeutic gene.

[0179] E22. A method for preparing a composition comprising a non-replicating HSV-based vector, comprising passaging an HSV-based vector comprising a transgene at least twice with immune serum having elevated levels of anti-HSV antibodies.

[0180] E23. The method of embodiment E22, wherein the HSV-based vector is an HSV-1 or HSV-2 vector.

[0181] E24. The method of embodiment E22 or E23, wherein the HSV-based vector has a membrane envelope comprising glycoproteins, at least one of which comprises the extracellular CD47 domain inserted at the N-terminus of the glycoprotein.

[0182] The method of embodiment E24, wherein the glycoprotein is selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L.

[0183] E26. The method of embodiment E25, wherein said glycoprotein is glycoprotein C.

[0184] E27. The method of any one of embodiments E22-E26, wherein said passaging in said immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of said HSV-based vector.

[0185] E28. The method of any one of embodiments E24-E27, wherein said extracellular domain comprises amino acids 19-141 of mouse CD47.

[0186] E29. The method of any one of embodiments E24-E27, wherein said extracellular CD47 domain is an extracellular human CD47 domain.

[0187] E30. The method of any one of embodiments E24-E29, wherein said HSV-based vector is free or substantially free of gE.

[0188] E31. The method of any one of embodiments E22-E30, wherein said immune serum comprises mammalian anti-HSV antibodies.

[0189] E32. The method of any one of embodiments E22-E31, wherein the immune serum is a mixture comprising rat serum and human serum having elevated levels of anti-HSV antibodies.

[0190] E33. The method of any one of embodiments E22-E32, wherein the method comprises passaging the HSV-based vector at least twice in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by at least one passaging in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

[0191] E34. A method of treating a disease in a patient in need thereof, comprising administering to said patient an HSV-based vector described in any one of embodiments E1-E7 or a composition described in any one of embodiments E8-E21.

[0192] E35. The method of embodiment E34, wherein the composition or HSV vector is administered systemically, intramuscularly, intradermally, or subcutaneously, or by intracerebroventricular or intraperitoneal injection.

[0193] E36. The method of embodiment E34 or E35, wherein the composition comprises cerebrospinal fluid.

[0194] E37. The method of any one of embodiments E34-E36, wherein the disease is a neurological disease.

[0195] E38. The method of any one of embodiments E34-E37, wherein the patient has been exposed to, or vaccinated against, HSV-1 and / or HSV-2, or has HSV-1 and / or HSV-2.

[0196] E39. A method of vaccinating a patient against a malignant or infectious disease comprising administering to said patient an HSV-based vector of any one of embodiments E1-E7 or a composition of any one of embodiments E8-E21.

Claims

1. A composition comprising a non-replicating herpes simplex virus (HSV)-based gene delivery vector, wherein the HSV vector comprises a transgene, and (a) the HSV vector has a membrane envelope comprising glycoproteins, at least one of which comprises an extracellular CD47 domain inserted at the N-terminus of the glycoprotein, (b) the HSV-based vector is prepared by passaging the HSV-based vector at least twice with immune serum having elevated levels of anti-HSV antibodies, or (c) both.

2. The composition of claim 1 , wherein the HSV vector is an HSV-1 or HSV-2 vector.

3. The composition of claim 1 or 2, wherein the HSV-based vector is an HSV amplicon packaged into a viral particle.

4. The composition of claim 1 , wherein at least one of the glycoproteins comprises an extracellular CD47 domain inserted at the N-terminus of the glycoprotein.

5. The composition of claim 4, wherein the extracellular CD47 domain is an extracellular human CD47 domain.

6. The composition of claim 1 , wherein the glycoprotein is selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L.

7. The composition of claim 6 , wherein the glycoprotein is glycoprotein C.

8. The HSV-based vector of claim 1 , wherein the transgene is a therapeutic gene.

9. 2. The composition of any one of the preceding claims, wherein the HSV vector is prepared by passaging the HSV vector at least twice with immune serum having elevated levels of anti-HSV antibodies.

10. 10. The composition of claim 9, wherein the passaging in the immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of the HSV-based vector.

11. 10. The composition of claim 9, wherein the HSV-based vector is free or substantially free of gE.

12. 10. The composition of claim 9, wherein the immune serum is a mixture comprising rat serum and human serum having elevated levels of anti-HSV antibodies.

13. 10. The composition of claim 9, wherein the HSV-based vector is prepared by passaging an HSV vector at least twice in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by at least twice in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

14. 10. A method for preparing the composition of claim 1, comprising passaging an HSV-based vector containing a transgene at least twice with immune serum having elevated levels of anti-HSV antibodies.

15. 13. A method of treating a disease in a patient in need thereof, comprising administering to said patient a composition according to claim 1.

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