Modified HSV-1 vector for heterogeneous expressions of transgenes

Abstract

The present invention is directed to a modified HSV-1 vector comprising at least two transgenes that exhibit controllable and distinct kinetics of expression, a pharmaceutical composition thereof suitable for use therapeutically or for in vitro applications, and methods relating thereto.

Description

[0001] TITLE: MODIFIED HSV-1 VECTOR FOR HETEROGENEOUS EXPRESSIONS OF TRANSGENES

[0002] BACKGROUND

[0003] During the last decade, viral delivery systems for gene delivery have seen a wave of improvement in introducing, replacing, or counteracting genes to treat a variety of human diseases. Adeno-associated virus (AAV) represents the leading platform for gene delivery, but such vectors are not adapted for the expression of large and / or multiple transgenes due to their small genome size, with a maximum transgene size of 4.7 kilobase pairs (kbp). While current herpes simplex virus type 1 (HSV-1) vectors offer alternatives for expressing large transgenes or multiple copies of the same transgene, viral vectors bearing multiple transgenes with distinct and controlled kinetics of expression are missing.

[0004] Various viral vector strategies have been developed. For example, polycistronic mRNA allows to produce multiple protein products from the same single mRNA (Wang et al., “Synthetic polycistronic sequences in eukaryotes” doi: 10.1016 / j.synbio.2021.09.003). However, with this strategy, the resulting proteins share the same stoichiometry and kinetic profile of expression. In addition, viral gene delivery systems have been generated with expression of several transgenes from distinct promoters being all inserted in a unique locus (Yu et al., “Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells,” doi.org / 10.1016 / S1525- 0016(03)00104-7), including oncolytic HSV-1 vectors (Haines et al., “ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity,” doi: 10.1158 / 2326- 6066.CIR-20-0609). As a result, although driven by different promoters, the transgenes displayed similar epigenetic regulation, thereby exhibiting common kinetic profiles of expression.

[0005] Therefore, there is a need to develop a novel viral vector that allows distinct epigenetic regulation to allow differentiated expression kinetics of different transgenes to respond to a diversity of innovative therapeutic strategies.

[0006] SUMMARY OF THE INVENTION

[0007] This invention provides for the first time a viral vector that allows different gene products (e.g., a protein) to be expressed from a single viral vector for different durations, which can be used for the treatment of various diseases, including genetic disorders, neurological disorders, and cancers.

[0008] Viral vectors of the invention enable specific kinetics of expressions of specific transgenes invite innovative therapeutic strategies. Using such novel viral vectors, a variety of transgenes can be delivered to the same cell and expressed for different durations. This application is based on the unexpected discovery that, by devising a novel viral vector comprising multiple transgenes located in selectively different regions along the vector genome, transgenes exhibit controllably distinct kinetic profiles of expression from each other, even delivered in the same cell. This novel viral vector opens the door for versatile gene therapies targeting a broad spectrum of diseases. The novel viral vector can be designed to deliver multiple transgenes to a cell, such as a neuron, an epithelial cell, a muscle cell, a connective tissue cell, or a platelet, preferably a neuron.

[0009] This novel viral vector is a modified Herpes Simplex Virus type 1 (HSV-1) vector, preferably a non-replicative HSV-1 (nrHSV-1) vector. The modified HSV-1 vector comprises at least two nucleic acid sequences that are expressed with different kinetic profiles. In some embodiments, the modified HSV-1 vector comprises a first nucleic acid sequence that is expressed during a first period and a second nucleic acid sequence that is expressed during a second period, that may or may not overlap with the first period, after entering a cell. In some embodiments, the first period is a short expression period, such as a transient expression period, for example, ranging from hours to days or a few weeks, . In some embodiments, the second period lasts for a long term, such as more than four weeks, or a life-long period of the subject of the delivery.

[0010] The multiple nucleic acid sequences are located in different regions along the modified HSV-1 vector. In some embodiments, the first nucleic acid sequence is located between genes expressed only during the lytic cycle, such as an intergenic region in the unique long (UL) region or the unique short (US) region. In some embodiments, the second nucleic acid sequence is located in a region surrounded by chromatin insulators that allows long-term expression, such as the latency-associated transcript (LAT) region.

[0011] In other embodiments, the first nucleic acid sequence is located in a genic region, where said gene is only expressed during the lytic cycle. This invention provides for the first time a viral vector that allows different gene products (e.g., a protein) to be expressed from a single viral vector for different durations, which can be used for the treatment of various diseases, including genetic disorders, neurological disorders, and cancers.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene located in the unique long (UL) region between UL21 and UL22 genes. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0014] Figure 2 shows nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene located in the unique long region (UL) between UL26.5 and UL27 genes. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0015] Figure 3 shows nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene located in the unique long (UL) region between UL45 and UL46 genes. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0016] Figure 4 shows nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene located in the unique short (US) region between US1 and US2 genes. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0017] Figure 5 shows the nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene located in the region deleted for the joint region. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0018] Figure 6 shows the nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene located in the terminal repeat long (TLR) between ICP34.5 and ICPO genes. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0019] Figure 7 shows the nrHSV-1 genome in which all IE genes are deleted, mGreenLantern transgene is located in the LAT region and the mScarlet transgene is located in the unique long (UL) region between UL45 and UL46 genes. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0020] Figure 8 shows the nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene replacing US1 (ICP22) coding sequence and driven by US1 IE4 / 5 viral promoter in the Unique Short region. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included). Figure 9 shows the nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene replacing US 12 (ICP47) coding sequence and driven by ICP47 IE4 / 5 viral promoter in the Unique Short region. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0021] Figure 10 shows the nrHSV-1 genome with mGreenLantern transgene located in the LAT region and mScarlet transgene replacing RL2 (ICPO) coding sequence and driven by ICPO IE4 / 5 viral promoter in the terminal repeat long region. AJoint region spreads from ICP27 (UL54, included) to ICP22 (US1, not included).

[0022] Figure 11A and Figure 11B shows a time-course analysis of transgenes expression in vitro in iPSC-derived cortical glutamatergic neuronal cells infected with the viral construct of Figure 3 (also referred to herein as “construct 3”), which encodes mGreenLantern and mScarlet reporters inserted into the LAT and UL45-UL46 InterGenic Region (IGR), respectively. Figure 11A shows the time-course analysis of live fluorescence in cells infected with the viral construct of Figure 3 expressing mGreenLantern under the control of the EFla promoter (green channel) and mScarlet under the Ubiquitin C promoter (red channel), observed at 3-, 8-, and 21 -days postinfection (dpi), with non-infected cells serving as controls. Figure 11B shows the quantitative analysis of reporter gene expression by qRT-PCR in cells infected by the viral construct of Figure 3. Statistical significance at each time point was assessed using the Mann-Whitney test, with p-values indicated as follows: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

[0023] Figure 12A and Figure 12B: shows a time-course analysis of transgenes expression in vitro in iPSC-derived cortical glutamatergic neurons infected with the viral construct of Figure 4 (also referred to herein as “construct 4”), which encodes mGreenLantern and mScarlet reporters inserted into the LAT and US1-US2 InterGenic Region, respectively. Figure 12A reveals robust and widespread mGreenLantern expression in infected iPSC-derived glutamatergic neurons at both 3 and 8 dpi, as determined by live-cell fluorescence microscopy. Figure 12B shows that quantitative qRT-PCR analysis supports the microscopy findings. Statistical significance at each time point was assessed using the Mann-Whitney test, with p-values indicated as follows: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

[0024] Figure 13 depicts qRT-PCR data quantifying mGreen Lantern and mScarlet reporter genes expression in iPSC-derived dopaminergic neurons infected with the nrHSV-1 vector of Figure 3 at 3 and 17dpi. Figure 14A and Figure 14B depict the percentage of positive animals for transgene expression in dorsal root ganglia (DRGs) of Balb / c mice over a 28-day period following injection.

[0025] Figure 14A shows that the percentage of animals exhibiting detectable mGreenLantern transgene expression by qRT-PCR increases over time following injection with the viral construct of Figure 3 (construct 3). Figure 14B shows the percentage of animals with detectable transgene expression in the L4-L6 DRGs following injection with the viral construct of Figure 4 (construct 4), as measured by qRT-PCR.

[0026] Figure 15A through Figure 15F show the kinetics and magnitude of mGreenLantern and mScarlet transgene expression over time in DRGs from Balb / c mice following footpad injection of HSV-1 -derived vectors of Figure 3 (construct 3) or of Figure 4 (construct 4). Statistical significance at each time point was assessed using the Mann-Whitney test, with p-values indicated as follows: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. Figures 15 A, B, and C correspond to animals injected with the vector of Figure 3, in which mGreenLantern is inserted into the LAT locus and mScarlet into the UL45-UL46 intergenic region. Figure 15A shows the expression kinetics of both reporters. mGreenLantern expression (black circle) is detectable at 3 dpi in the majority of animals and increases steadily over time, reaching approximately 2000% of its initial value by 14 dpi and stabilizing to a plateau up to 28 dpi. In contrast, mScarlet (grey square) expression is minimal across the time course, becoming nearly undetectable in all tested animals from 14 dpi onwards. Figure 15B shows a box-and-whisker plot of mGreenLantern expression levels over time for animals injected with the viral vector of Figure 3, illustrating a progressive increase in median values from day 3 to day 28. Figure 15C displays the expression of mScarlet normalized to the level of mGreenLantern at day 3. mScarlet reaches approximately 16% of the mGreenLantern reference at early time points (3 dpi) but falls to negligible levels by 7- to 14-dpi. Figures 15D, E, and F present data from animals injected with the vector of Figure 4 in which mGreenLantern is inserted into the LAT locus and mScarlet into the US1-US2 intergenic region. Figure 15D indicates that mGreenLantern expression (black circle) is significantly higher than mScarlet at all time points, reaches a maximum of approximately 450% by day 14, relative to day 3. In contrast, mScarlet (grey square) expression is initially low and decreases rapidly, becoming nearly undetectable from 21 dpi onwards in all tested animals. Figure 15E illustrates mGreenLantern expression kinetic over time in animals injected with vector of Figure 4, showing increase from day 3 to day 14 and then plateau up to 28dpi. Figure 15F shows mScarlet expression as a percentage of the mGreenLantern day 3 baseline. A small transient expression is observed at 3 dpi in 7 out of 8 animals, mScarlet levels decline rapidly at day 7 and remain close to background from 14 dpi onward. Notably, mScarlet expression levels in animals injected with vector of Figure 4 are slightly higher and persist slightly longer than in animals injected with vector of Figure 3, suggesting that positioning mScarlet in the US1-US2 intergenic region may delay, but not prevent, the onset of transcriptional silencing.

[0027] DETAILED DESCRIPTION

[0028] Herpes simplex virus (HSV) is a complex, non- integrating DNA virus capable of infecting a very wide range of human and animal cells. HSV encompasses two serotypes, herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). The genome of HSV- 1 has a size of approximately 152-kbp. It contains about 80 protein- encoding genes and more than 20 microRNA. The HSV-1 genome is composed of two unique segments, UL and US, each flanked by inverted repeats that encode critical genes present in two copies.

[0029] This invention is based on the unexpected discovery that two or more transgenes delivered by a modified HSV-1 vector can exhibit distinct and controllable kinetic profiles of expression after entering a target cell. This modified HSV-1 vector offers unique advantages over other viral vectors in gene therapy, paving the way for innovative therapeutic strategies that are currently unavailable. The Applicant has discovered that the insertion of multiple transgenes respectively into different, selected regions of the HSV-1 genome can provide distinct kinetic profiles of expression for each transgene in various cell types. In the simplest form of this invention, two transgenes are independently inserted into the HSV-1 vector wherein a first transgene is inserted into a region of the genome configured to confer short-term (e.g., transient) expression and a second transgene is inserted into a region of the genome configured to confer long-term expression of the transgene. For example, in embodiments, the first transgene is inserted into a short-expression region, such as between genes expressed only during the lytic cycle (e.g., in an intergenic region in the UL or US region, preferably between lytic genes) of the genome, and the second transgene surrounded by chromatin insulators (e.g., inserted into the LAT region) of the genome. In embodiments, the first transgene expresses a gene product (e.g., a protein) for a first period and the second transgene expresses a gene product for a second period. The first and second periods may or may not overlap with each other.

[0030] Preferably, the modified HSV-1 vector is a non-replicative HSV-1 vector (nrHSV-1). nrHSV-1 vectors are engineered to lack essential viral genes required for replication, typically the immediate-early genes such as ICP4 (both copies), ICP27, and ICPO (one or both copies). These vectors can express transgenes but cannot produce new virions.

[0031] A wild-type genome of HSV-1 has a size of approximately 152 kbp and comprises some 80 protein-encoding genes and at least 20 microRNA. However, a large part of this genome encodes non-essential genes that could, in principle, be individually deleted without significantly perturbing virus multiplication and packaging in cultured cells. In addition, one or more essential genes can also be deleted, but in this case the virus will only multiply in the presence of a complementing system, such as a complementing cell line, that provides the proteins not expressed by the vector genome. Therefore, the genome of HSV-1 can be modified such that non-essential genes, essential genes, or combinations thereof can be deleted. Altogether, these deletions can create a vast genomic space allowing the introduction and delivery of very large foreign pieces of DNA.

[0032] In some embodiments, the modified HSV-1 vector comprises a HSV-1 pre- vector, wherein non-essential genes, essential genes, or combinations thereof have been deleted from the HSV-1 genome to arrive at a genome backbone comprising less than 130 kbp and greater than 75 kbp. HSV-1 pre-vectors embodying the invention are described with the understanding that, as "backbones," it is contemplated that one or more nucleic acids, with or without extraneous control elements, can be inserted therein.

[0033] According to the invention, the qualifier “essential” in the expression “essential genes” or “non-essential genes”, means that the given gene is essential (or not) for achieving multiplication and packaging of the virus genome, thus generating infectious progeny virus particles. HSV-1 essential genes include ULI, UL5-UL9, UL12, UL14, UL15, UL17-UL19, UL22, UL25-UL38, UL42, UL48, UL49, UL52, UL54, US6, ICP4 (2 copies). HSV-1 non-essential genes include ICP34.5 (2 copies), ICPO (2 copies), LAT (2 copies), UL2-UL4, UL10, UL11, UL13, UL16, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43-UL47, UL50, UL51, UL53, UL55, UL56, US1-US5, US7-US12. In some embodiments, clusters of genes that could be deleted include, but are not limited to, genes UL2, UL3, UL4 (10.200 - 12.600); genes UL10, ULI 1 (23.200 - 25.200); gene UL16 (30.200 - 31.400); genes UL20, UL21 (40.800 - 43.700); genes UL23, UL24 (46.700 - 48.600); genes UL39, UL40, UL41 (86.400 -92.700); genes UL43 to UL47 (94.700 - 103.200); genes UL50, UL51 (107.700 - 109.100); genes UL55, UL56 (115.400 - 117.100); one copy of genes LAT, ICP0, UL34.5 (IRL) (118.700 - 126.100); genes US2 to US5 (134.000 - 138.200); genes US7 to US12 (139.700 - 145.600); and / or the second copy of gene ICP0 when the first copy has already been removed among the LAT, ICP0, UL34.5 cluster.

[0034] In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 22 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 25 kbp have been deleted. In some embodiments, the HSV-1 pre- vector comprises a genome wherein at least 30 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 40 kbp have been deleted. In some embodiments, the HSV- 1 pre-vector comprises a genome wherein at least 45 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 50 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 55 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 60 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein at least 65 kbp have been deleted. In some embodiments, the HSV- 1 pre-vector comprises a genome wherein at least 75 kbp have been deleted.

[0035] In some embodiments, the HSV-1 pre-vector comprises a genome wherein 25 kbp to 80 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein 30 kbp to 75 kbp have been deleted. In some embodiments, the HSV-1 pre-vector comprises a genome wherein 35 kbp to 70 kbp have been deleted. In some embodiments, the HSV-1 pre- vector comprises a genome wherein 40 kbp to 60 kbp have been deleted.

[0036] In some embodiments, the modified HSV-1 vector comprises a HSV-1 pre- vector comprising an HSV-1 genome wherein non-essential genes, essential genes, or combinations thereof have been deleted to arrive at a genome backbone comprising less than 130 kbp and greater than 75 kbp.

[0037] In some embodiments, the HSV-1 pre-vector further optionally comprises a Bacterial Artificial Chromosome (BAC) sequence. As used herein, a BAC is an engineered DNA molecule used in a fashion that allows it to be propagated as a circular artificial chromosome in bacteria. BAC vectors are plasmids constructed with the replication origin of E. coli F factor, and so can be maintained in a single copy per cell. These BAC vectors can hold DNA fragments of up to 300 kbp. Although all non-essential HSV-1 genes can in principle be deleted within a BAC, which is propagated in bacteria, the collective deletion of all of them will most probably create a disabled virus, too much attenuated to be efficiently grown in mammalian cultured cells.

[0038] Preferably, the HSV-1 pre-vectors used in this invention maintain enough of the HSV-1 genome so as to not become HSV-1 amplicon. By "Amplicon or amplicon vector," it is meant to be a helper-dependent vector, the genome of which lacks most or all HSV genes coding for viral proteins. The genome of amplicon vectors is a concatemeric DNA composed of multiple copies, organized in tandem, of a plasmid-known as the amplicon plasmid- that carries one origin of DNA replication and one packaging signal from HSV-1 genome. In cells expressing the full set of structural, replication and DNA packaging functions from HSV-1, resulting from the presence of an HSV-1 genome acting as helper, the amplicon plasmid is amplified by a rolling-circle mechanism into long head-to-tail concatemers that are then cleaved and packaged, up to one genome size, into HSV-1 virions (Kwong and Frenkel, 1985; Bataille and Epstein, 1997). Amplicon vectors are thus concatemeric DNA sequences packaged into HSV-1 particles.

[0039] The HSV-1 pre-vector serves as a vector template, wherein deletions described above create genomic space allowing the introduction of one or more nucleic acid sequences of interest. The introduction of multiple nucleic acid sequences, e.g., a transgene of interest, increases the genome size (i.e., number of base pairs) in the resulting modified HSV-1 vector of the invention. Without wishing to be bound to any particular theory, it is believed that when the modified HSV- 1 vector of the invention is of a size similar to the 152 kbp of the wild-type HSV-1 genome or within a range of 143kbp to 154.5 kbp, the resulting modified HSV-1 vector is more stable genomically and, thereby possessing improved vector viability.

[0040] In any embodiment herein, the size of the modified HSV-1 vector is between about 143 kbp and 160 kbp. In some embodiments, the size of the modified HSV-1 vector is between about 143 kbp and 158 kbp. In some embodiments, the size of the modified HSV-1 vector is between about 143 kbp and 155 kbp. In some embodiments, the size of the modified HSV-1 vector, is between about 143 kbp and 153 kbp. In any embodiment herein, the size of the modified HSV-1 vector is between about 147 kbp and 165 kbp. In some embodiments, the size of the modified HSV-1 vector is between about 147 kbp and 153 kbp. In some embodiments, the size of the modified HSV-1 vector is between about 149 kbp and 155 kbp. In some embodiments, the size of the modified HSV-1 vector, is between about 149 kbp and 153 kbp. In some embodiments, the size of the modified HSV-1 vector, is between about 152 kbp.

[0041] The modified HSV-1 vector comprises at least two inserted nucleic acid sequences, each independently encoding a transgene of interest. The term "transgene" refers to a particular nucleic acid sequence, including coding sequence for a gene product or non-coding sequence for an RNA product to be expressed in a cell into which the nucleic acid sequence is introduced.

[0042] A “gene product” is a molecule resulting from the expression of a particular gene. A “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product after being transcribed, and sometimes also translated. In some instances, the gene consists or consists essentially of coding sequence, that is, sequence that encodes the gene product. In other instances, the gene comprises additional, non-coding, sequence. For example, the gene may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons). Gene products include, e.g., a protein, a polypeptide, a peptide, an aptamer, an mRNA, and the like.

[0043] A “RNA product” is a sequence that is transcribed into a non-coding nucleotide sequence, such as an interfering RNA including short interfering RNA (siRNA), microRNA (miRNA), small hairpin RNA (shRNA), antisense oligonucleotide (asRNA), and the like. Such RNA products can silence a specific gene and / or to disrupt the corresponding encoded protein (a “gene of interest” or “targeted gene” or “selected gene”). By “silencing” a gene, it is meant that expression of the target gene is reduced or eliminated. Without being bound by theory, it is believed that silencing is characterized by specific mRNA degradation or mRNA block in translation after the expression of a non-coding complementary sequence such as siRNA, asRNA, shRNA, miRNA, or any other form of interfering RNA (iRNA) into cells.

[0044] The at least two inserted nucleic acid sequences each independently encode a gene product or an RNA product as defined above. In some cases, the at least two inserted nucleic acid sequences each independently encode a gene product as defined above. In some other cases, the at least two inserted nucleic acid sequences each independently encode a RNA product as defined above. In additional cases, the at least two inserted nucleic acid sequences each independently encode a gene product or a RNA product as defined above, provided that at least one inserted nucleic acid sequence encodes a gene product and at least one inserted nucleic acid sequence encodes a RNA product. In some embodiments, a gene product comprises a therapeutic gene product, e.g., a therapeutic protein. In some embodiments, at least one gene product encoded by the inserted nucleic acid sequence is a therapeutic gene product, e.g., a therapeutic protein.

[0045] The term "transgene" includes (1) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been introduced; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been introduced. By "mutant form" is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that the transgene product will be secreted from the cell, or the transgene may include both a leader peptide or signal sequence plus a membrane anchor peptide, or even be a fusion protein between two naturally occurring proteins or part of them, such that the transgene will remain anchored to cell membranes, or a sequence that allows the protein to accumulate in a specific region of the cell, such as a nuclear localizing signal. As used herein, the term “transgene,” when appearing alone without “first” or “second,” includes both the first transgene and the second transgene.

[0046] In some embodiments, the modified HSV-1 vector comprises at least two inserted nucleic acid sequences, each encoding a gene product, and optionally one or more staffers. The size of the modified HSV-1 vector genome is about 143 kbp to about 154.5 kbp. The size of the HSV-1 pre-vector genome is itself important, as a genome that is too short or too large cannot be correctly packaged and requires compensation for the size. The presence and / or length of a stuffer is tailored according to the inserted nucleic acid sequences to arrive at the desirable overall length of the modified HSV-1 vector genome.

[0047] The inserted multiple nucleic acid sequences include DNA. In any embodiment disclosed herein, the inserted multiple nucleic acid sequences comprise DNA. In any embodiment disclosed herein, the inserted multiple nucleic acid sequences comprise combinations of coding or non-coding DNA.

[0048] Preferably, at least one of the inserted multiple nucleic acid sequences encodes a protein of interest, such as a therapeutic protein. In some cases, at least one of the inserted multiple nucleic acid sequences encodes a non-coding RNA. In some cases, at least two of the inserted multiple nucleic acid sequences each independently encode a protein of interest, such as a therapeutic protein.

[0049] In any embodiment disclosed herein, at least two of the inserted multiple nucleic acid sequences each independently encode a transgene of interest. In additional preferred embodiments, at least one of the inserted multiple nucleic acid sequences independently encodes a non-coding RNA. In any embodiment disclosed herein, the multiple nucleic acid sequences can be introduced into the HSV-1 pre-vector backbone by site specific recombination (SSR), by homologous recombination (HR), or En passant mutagenesis technique. In any embodiment disclosed herein, the multiple nucleic acid sequences are introduced by homologous recombination or En passant mutagenesis technique.

[0050] Non-limiting examples of transcribed sequences that can be present in a transgene within the inventive vector include, but are not limited to, a sequence expressing a long transgene, a sequence expressing a transgene able to generate different splice variants, a sequence expressing two or more transgenes, either as a single transcription unit or in separated transcription units, a sequence expressing a transgene under the control of very long upstream and / or downstream regulatory sequences, a sequence expressing short regulatory RNAs (miRNA, siRNA, etc.), a sequence expressing long non-coding RNAs (IncRNA), a sequence conferring the ability to be replicated and correctly segregated in dividing cells, or combinations of the above sequences.

[0051] The transgenes of the invention independently include one or more transcribed sequence(s), which are expressed under the control of the promoter and optionally other regulatory elements within the transgene. A transcribed sequence can be any sequence desired to be expressed within a given cell into which the vector is to be introduced. Non-limiting examples of transcribed sequences that can be present in a transgene within the inventive vector include Oct4, Klf4, Sox2, c-Myc, L- myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, GATA4, Nkx2.5, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3, SERCA2a, Pax3, MyoD, Lhx2, FoxGl, FoxP2, Isll , Ctip2, Tbrl , Ebfl, Gsx2, Srebp2, Factor VIII, Factor IX, Dystrophin, CFTR, GlyRal, enkephalin, GAD67 (or other GAD isoforms, e.g., GAD 65), TNF , IL-2, IL-4, IL- 10, IL- 12, GM-CSF, a neurotrophic factor (e.g., NGF, BDNF, GDNF, NT-3), Ascii, Nurrl, LmxlA, Brn2, Myth, NeuroDl, FoxA2, Hnf4a, Foxal , Foxa2 or Foxa3, any microRNA or combination of miRNAs (e.g., hsa-mir-302 / 367 gene cluster; hsa-miR200c; hsa-miR369; hsa- mir-124) and / or one or more other non-coding RNAs ("ncRNA(s)") or a reporter gene for expression in mammalian cells, such as LacZ (encoding beta-galactosidase), CAT (encoding chloramphenicol acetyltransferase), a luminescent reporter gene (e.g. firefly luciferase), or a fluorescent protein-encoding gene (e.g., GFP, YFP, RJFP, and analogues thereof such as iRFP, EGFP, and the like).

[0052] In some embodiments, this invention utilizes the heterogenous expression kinetic of HSV-1 in cells to allow for control and distinct expression profiles for at least two inserted nucleic acid sequences that encode gene products.

[0053] The wild-type HSV-1 follows a well-defined sequence of events from initial infection to the production of new virions. During the entry step, which occurs within 0-1 hour postinfection, HSV-1 virions bind to heparan sulfate proteoglycans and interact with specific receptors such as nectin-1. The viral envelope fuses with the cell membrane, allowing the nucleocapsid and tegument proteins to enter the cytoplasm. The nucleocapsid is then transported along microtubules to nuclear pores, releasing viral DNA into the nucleus. Between 2-4 hours post-infection, the immediate-early (IE) phase begins, with the viral DNA undergoing transcription to produce IE proteins such as ICPO, ICP4, ICP22, ICP27, and ICP47. These regulatory proteins modulate the host cell environment to favor viral replication. From 4-8 hours post-infection, the early (E) phase ensues, with IE proteins activating the transcription of early genes that encode enzymes necessary for viral DNA synthesis, such as DNA polymerase and thymidine kinase. During this period, viral DNA replication begins, increasing viral DNA copies for the production of late genes. The late (L) phase, spanning 8-16 hours post- infection, involves the transcription of late genes that encode structural proteins required for assembling new virions. The assembly and egress phase, from 16-24 hours post-infection, leads to the assembly of new viral capsids in the nucleus, the encapsidation of replicated viral DNA and the acquisition of their final envelope by budding into cytoplasmic vesicles. Mature virions are released from the cell by exocytosis or cell lysis. The release of progeny virions, occurring from 24-48 hours post-infection, spreads the infection to neighboring cells. nrHSV-1 vectors, lacking essential viral genes required for replication, are unable to replicate nor induce a lytic phase but have the ability to express a transgene. Without wishing to be bound by any particular theory, in neuronal cells, where HSV-1 establishes latency, a transgene inserted between lytic genes can be expressed for a short period of time before its heterochromatinization, while a transgene inserted in a region flanked by chromatin insulators (such as the LAT region) can be expressed for the long term. Similarly, in non-neuronal cells, as non-replicative vectors fail to launch a lytic phase due to the deletion of essential genes, the viral genome can enter in a latent state after this failure. Hence, a transgene inserted between lytic genes should be expressed for a short time before repression, while a transgene inserted in the insulator-protected region should be expressed for the long term.

[0054] The modified HSV-1 vector comprises at least two transgenes inserted within different regions of the modified HSV-1 vector to provide distinct kinetic profiles of expressions, wherein at least a first transgene exhibits a first kinetic profile of expression and at least a second transgene exhibits a second kinetic profile of expression after entering a cell, wherein the first kinetic profile and the second kinetic profile are different from each other.

[0055] The modified HSV-1 vector comprises at least two transgenes inserted within different regions of the modified HSV-1 vector to provide distinct kinetic profiles of expressions, wherein at least a first transgene is expressed for a first period and at least a second transgene is expressed for a second period after entering a cell, and wherein the first period is a shorter period than the second period.

[0056] In some cases, the first period and the second period begin at about the same time.

[0057] In some cases, the first period begins before the second period.

[0058] In some cases, the second period begins after the end of the first period. In some cases, the second period begins after the beginning of the first period and before the end of the first period.

[0059] In some embodiments, the first period is transient, i.e., the first transgene is characterized by a transient expression. As used herein, the term “transient” is opposite to the persistent or long-term expression and refers to a temporary expression that will be terminated, preferably by constitutive and / or facultative heterochromatinization.

[0060] In some embodiments, the first period lasts no more than about four weeks. In some preferred embodiments, the first period ranges from about one hour to about three weeks. In preferred embodiments, the first period ranges from about two hours to about two weeks. In even preferred embodiments, the first period ranges from about three hours to about one week.

[0061] In preferred embodiments, the first period is within about four weeks. In some embodiments, the first period is within about three weeks. In preferred embodiments, the first period is within about two weeks. In preferred embodiments, the first period is within about one week. In preferred embodiments, the first period is within about 6 days, about 5 days, about 4 days, or about 3 days.

[0062] In some embodiments, the second period is for a long term, i.e., the second transgene is characterized by a long-term expression.

[0063] In some embodiments, the second period lasts no less than about four weeks.

[0064] In some embodiments, the second period lasts about one month, three months, six months, one year, two years, three years, or longer than three years. For example, the inventive HSV1 vector can express a transgene for at least 28 days, and preferably for at least 60 days. In some embodiments, the second period lasts about 30 to 45 days, or from 45 to 90 days, or from 90 to 365 days, or 365 days to several years. In some embodiments, the second period lasts for a life or nearly a life time of the subject to whom the vector is administered. As used herein, the term “life time” or “lifelong” refers to a length of time starting from the beginning of the expression of the second transgene to the death of the subject to whom the vector is administered.

[0065] In some embodiments, the first period is at least about 2 hours shorter than the second period. In some embodiments, the first period is at least about 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 hours shorter than the second period. In some embodiments, the first period is at least about 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, one month, two months, three months, six months shorter than the second period. In some embodiments, the first period is at least about one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, ten years shorter than the second period. The second transgene is operably linked to at least one sequence conferring long-term expression. In some embodiments, the second transgene is introduced downstream of a chromatin insulator. In some embodiments, the second transgene is introduced between two chromatin insulators.

[0066] In some embodiments, the second transgene can be introduced in a LAT region. A LAT (Latency Associated Transcripts) locus or region is a repeated locus that is contained in the inverted repeated sequences known as b and b' of the virus genome. The b and b' sequences of the virus genome are also known as TRL (Terminal Repeat Long) and IRL (Internal Repeat Long), respectively. In some embodiments, the HSV-1 vector genome contains both LAT regions, one in the TRL and the other in the IRL. In some embodiments, one of the LAT region, either in the TRL or in the IRL, has been deleted. In some embodiments, when the HSV-1 prevector genome contains the two LAT loci, the second transgene can be introduced into both loci, in the TRL region and in the IRL region. In some embodiments, when the LAT locus in the IRL region is deleted, the second transgene can be introduced into the LAT locus in the TRL region only. In some embodiments, when the LAT locus in the TRL region is deleted, the second transgene can be introduced into the LAT locus in the IRL region only. Preferably, the second transgene can be introduced into the LAT locus in the TRL region. More preferably, the LAT locus in the IRL region is deleted, and the second transgene can be introduced into the LAT locus in the TRL region only, as shown in Figures 1-6.

[0067] The LAT locus includes an upstream DNA insulator (INS or CTRL1) sequence, the Latency Associated Promoter (LAP), a region conferring Long-Term Expression (LTE) and a downstream DNA insulator (INS). In some embodiments, the second transgene is introduced either between the Latency Associated Promoter (LAP) and the Long-Term Expression (LTE) region, or between the LTE region and the DNA insulator (INS or CTRL2) sequence present downstream of the LTE.

[0068] Importantly, the LAT locus contains both the LTE and the DNA insulator sequences (INS) that confer long-term expression to the second transgene, e.g., a therapeutic transgene, introduced into this site. In some embodiments, the second transgene will be localized between the long-term expression (LTE) and the downstream DNA insulating (INS) motifs.

[0069] By "long-term expression sequence" or "long-term expression element (LTE)" it is meant a nucleotide sequence that when operably linked to a foreign DNA of interest (e.g., the second transgene) allows for sustained expression of a gene product for about 28 to 45 days, or from 45 to 90 days, or from 90 to 365 days, or 365 days to several years or even during the life of the patient.

[0070] Long-term expression (LTE) sequences were identified in HSV-1 as a region of the latency-associated transcripts (LAT), which originate from the LAT-associated promoter (LAP). This LTE is located downstream of the LAT transcription start site. Preferably, the LTE is comprised between about 1.5 kb to about 3 kb downstream of the LAT transcription start site. Additionally, chromatin insulators (also called DNA insulators) also contribute to providing long-term expression. Without wishing to be bound to any particular theory, DNA insulators may inhibit epigenetic silencing. Sequences conferring long-term expression (both the LTE and the DNA insulator sequences) can be placed either upstream and / or downstream the foreign DNA.

[0071] The LAT locus is surrounded by chromatin insulators, favorizing its expression compared to the repressed lytic genes (Washington et al., “CTCF Binding Sites in the Herpes Simplex Virus 1 Genome Display Site-Specific CTCF Occupation, Protein Recruitment, and Insulator Function,” doi: 10.1128 / JVI.00156-18). Chromatin insulators can decrease the risk of insertional mutagenesis by disrupting the interactions between the enhancers in the vectors and the regulatory elements of cellular oncogenes. There are two kinds of chromatin insulators: barrier insulators, which protect chromosomal domains from heterochromatinization, and enhancerblocking insulators, which prevent the interaction between regulatory elements of different chromatin domains. Certain elements combine barrier- and enhancer-blocking activities. In some embodiments, the second transgene is introduced downstream of one chromatin insulator. In some embodiments, the second transgene is introduced between a pair of chromatin insulators. Figures 1-6 illustrate mGreenlantem reporter transgene, as the second transgene described herein, is inserted into the LAT region between two chromatin insulators (CTRL2 and CTRL1) to convey long-term expression.

[0072] Those skilled in the art will recognize that other LTE-like sequences, as well as other DNA insulator sequences, have been described and are continually being discovered. For example, the ICP4 locus is also surrounded by CTCF-dependent chromatin insulators. A cluster of CTCF motifs called CTRS3 is located at the 5’ of the ICP4 locus and three cluster of CTCF motifs, CTRS1, CTRS2, and Cta'm, are located at its 3’ end (Amelio et al., 10.1128 / JVI.80.5.2358-2368.2006, Bloom et al., 10.1016 / j.bbagrm.2009.12.001). All such LTE- like sequences and DNA insulator sequences are encompassed by the present invention.

[0073] In any embodiment disclosed herein, the first transgene is introduced into the HSV-1 vector between its lytic genes, thereby being preferably expressed before the heterochromatinization. In some preferred embodiments, the first transgene is only expressed before the heterochromatinization. Namely, the first period as described above preferably ends before the heterochromatinization.

[0074] Heterochromatinization of the genomic regions containing the transposon- retrotransposon sequences and other repeated sequences is a strategy developed against them. Such heterochromatinic regions are commonly called constitutive heterochromatin. Facultative heterochromatinization refers to the heterochromatinization of the particular gene domains during cellular differentiation.

[0075] Facultative heterochromatin refers to chromatin regions that can switch between condensed, transcriptionally silent states and more relaxed, transcriptionally active states depending on cellular context and environmental signals. In the context of HSV-1 vectors, facultative heterochromatin plays a crucial role in regulating viral gene expression, particularly during latency and reactivation. During latency, the HSV-1 genome becomes associated with both constitutive and facultative heterochromatins, leading to the silencing of most viral genes to maintain the dormant state. This involves histone modifications and the binding of repressive complexes, compacting the viral DNA and rendering it inactive. Reactivation from latency involves the disruption of heterochromatin, allowing the transcription of immediate-early genes (IE) and initiating the lytic cycle. Therefore, in the modified HSV-1 vectors, the first transgene inserted between lytic genes is expressed before the lytic genes become heterochromatinized and repressed. The lytic genes as used herein include IE genes (such as ICPO, ICP27), Early (E) genes (such as US1, US2, UL9, UL30, UL42, UL 23), and Late (L) genes (such as UL19, UL21, UL22, UL26, UL27, UL38, UL45, UL46, UL48).

[0076] In some embodiments, the first transgene is inserted between lytic genes in the UL region, US region, and / or TRL region. In some embodiments, the first transgene is inserted between lytic genes in the UL region and / or US region. In some embodiments, the first transgene is inserted between lytic genes in the UL region. In some embodiments, the first transgene is inserted between lytic genes in the US region. In additional embodiments, the first transgene is inserted between lytic genes in the TRL region.

[0077] In some embodiments, the first transgene is inserted into the intergenic region between two convergent lytic genes in the UL region or in the US region. In some embodiments, the first transgene is inserted into the intergenic region between two convergent lytic genes in the UL region, such as UL3-UL4, UL7-UL8, UL10.5-UL11, UL15-UL16, UL21-UL22, UL26.5-UL27, UL30-U131, U135-UL36, UL40-UL41, UL45-UL46, UL50-UL51, or UL55-UL56. For example, as illustrated in Figure 1-3 respectively, the first transgene (mScarlet reporter transgene) is inserted into the intergenic region between UL21 and UL22, UL26.5 and UL27, or UL45 and UL46. Without being bound by the theory, the first gene is preferably inserted into the intergenic region between convergent genes to avoid intervening with the promoter regions.

[0078] In some embodiments, the first transgene is inserted into the intergenic region between convergent lytic genes in the US region. In some embodiments, the first transgene is inserted into the intergenic region between convergent lytic genes in the US region, such as US1-US2, or US9-US10.

[0079] For example, as illustrated in Figure 4, the first transgene (mScarlet reporter transgene) is inserted into the intergenic region between US1 and US2.

[0080] In some embodiments, the first transgene is inserted into a region where the original gene has been deleted, as described herein. For example, the first transgene is inserted into a region deleted for the joint region, as shown in Figure 5.

[0081] In some embodiments, the first transgene is inserted into the intergenic region between ICP34.5 (i.e., y34.5) and ICPO in the TRL region, as shown in Figure 6.

[0082] In other embodiments, the first transgene (also referred to herein as the first nucleic acid sequence) is inserted in a genic region, where said gene is only expressed during the lytic cycle. In embodiments, the first nucleotide acid sequence is under the control of the natural viral promoter for the genic region where it is inserted.

[0083] In some embodiments, the first transgene is inserted in place of an IE gene region, under the control of the natural viral promoter for the IE gene where it is inserted. “In embodiments, the IE coding sequence gene is an IE coding sequence gene that is expressed during the lytic cycle.” For example, the first transgene is inserted downstream of the US1 promoter, US 12 promoter or the ICPO promoter and replaces the coding sequence of US 1, US 12, or ICPO. For example, as illustrated in Figures 7 to 9, the first transgene (mScarlet reporter transgene) is inserted downstream the US1 promoter, the US 12 promoter, or the ICPO promoter, respectively As used herein, although “the first transgene” and “the second transgene” are used in singular forms, they are both meant to include the plural form as well. The term “the first transgene” refers to one or more transgenes that are expressed for the first period as defined herein. The first transgene can be only one transgene, or multiple distinct transgenes, or multiple copies of the same transgene. The term “the second transgene” refers to one or more transgenes that are expressed for the second period as defined herein. Similarly, the second transgene can be only one transgene, or multiple distinct transgenes, or multiple copies of the same transgene.

[0084] In some embodiments, the second transgene (also referred to herein as the second nucleic acid sequence) is inserted in an operable connection with one or more LTEs and / or DNA insulator sequences within the modified HSV-1 vector. The first transgene is inserted in an operable connection with one or more lytic gene sequences within the modified HSV-1 vector. By "operably connected" or “in an operable connection” in regard to the second transgene, it is to be understood that the one or more LTEs and / or DNA insulator sequences allow the expression of the transgene in a cellular environment in which genetic elements (i.e., "genes") otherwise present within the HSV genome are preferably transcriptionally silent. By "operably connected" or “in an operable connection” in regard to the first transgene, it is to be understood that the first transgene is expressed for a certain period of time, preferably before heterochromatinization, in a cellular environment.

[0085] Within the transgene(s) inserted into the inventive vector, there is at least a promoter sequence and a transcribed sequence such that the transcribed sequence(s) is controlled by the promoter. A “promoter”, as used herein, is a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of an operably linked downstream (3' direction) sequence. For purposes of the present invention, a promoter sequence includes at least the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as RNA polymerase binding domains. Eukaryotic promoters will often, but not always, contain "TATA" boxes and other DNA motifs, such as "CAT" or "SP1" boxes.

[0086] The promoters for the first transgene and the second transgene can be independently any promoter desired to control / regulate the expression of the transcribed sequence(s). In some embodiments, the promoter within a transgene expression cassette inserted into the inventive vector can be a constitutive mammalian promoter, such as are known in the art (e.g., EFla, UbC, P-actin, PGK, U6 and the like).

[0087] In some embodiments, the promoter can be a cell-specific or tissue-specific promoter (e.g., EOS, OCT4, Nanog (for ESC / iPSC), SOX2 (for neural stem cells), aMHC, Brachyury, Tau, GFAP, NSE, Synapsin I (for neurons), Apo A-I, Albumin, ApoE (for liver), MCK, SMC a- Actin, Myosin heavy chain, Myosin light chain (for muscle), etc.), such as a promoter that specifically or preferentially control expression of genes in a defined cell type (e.g., within a liver cell, lung cell, epithelial cell, cardiac cell, neural cell, skeletal muscle cell, embryonic, induced pluripotent, or other stem cell, cancer cell, etc.).

[0088] In some embodiments, promoters for use in sensory neurons include promoters of genes coding for sensory neuroreceptors such as Transient Receptor Potential Vanilloid 1 (TRPV1) or Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), or from promoters of genes coding for sensory neuromodulators or sensory neurotransmitters, such as the promoters of Substance P, PACAP, Calcitonin Gene Related Peptide (CGRP). In embodiments, promoter of genes coding for sensory neuroreceptors according to the invention is a promoter of the TRP gene family, more preferentially the promoter TRPV1 or TRPM8. In embodiments, promoters of genes coding for sensory neuromodulators or sensory neurotransmitters according to the invention is the CGRP, or the promoter of genes involved in neurite outgrowth and stress response in sensory neurons, preferably the promoter of the gene encoding advillin (ADVL). In other embodiments, the promoter within a transgene inserted into the inventive vector can be an inducible promoter.

[0089] In some cases, promoters use for the first transgene and the second transgene are the same. In some cases, promoters for the first transgene and the second transgene are different. In some cases, wherein the first transgene includes more than one first transgene, the promoters for all the first transgenes can be independently any promoter desired to control / regulate the expression of the transcribed sequence(s). In other words, they can be independently identical or different among themselves. In some cases, wherein the second transgene includes more than one second transgene, the promoters for all the second transgenes can be independently any promoter desired to control / regulate the expression of the transcribed sequence(s). In other words, they can be independently identical or different among themselves. In any embodiment disclosed herein, the one or more transgenes can be part of one or more expression cassettes. The term "expression cassette" as used herein refers to any nucleic acid sequence containing a promoter and a downstream coding sequence or transgene, which expression is driven by said promoter, which is followed by a polyadenylation signal.

[0090] In addition to the promoter(s) and coding sequence(s), the transgene(s) inserted into the genome of the inventive vector also can comprise additional regulatory element(s). For example, the transgene(s) can include one or more sites for binding of microRNA. The presence of such sites facilitates down-regulation of the transgene expression in certain cell types. Thus, for example, a vector comprising a transgene desired to be expressed specifically in a cancer or tumor cell (which may be toxic to many cell types) can comprise binding sites for microRNAs of "normal" (i.e., non-malignant) cells, so that the expression of the transgene is suppressed in non- malignant cells.

[0091] In some embodiments, transgene(s) within the inventive vector can be monocistronic (i.e., encoding a single mRNA, resulting in a single protein or polypeptide) or polycistronic (i.e., encoding multiple mRNA, resulting in more than one protein or polypeptide) or can express a single or multiple mRNAs that encode self-cleavable polyproteins. In embodiments, all or part of the transcribed portion of the transgene also can encode non-translated RNA, such as siRNA or miRNA. In embodiments, the inventive vector can comprise multiple separate monocistronic or polycistronic transgene units, each with its own respective promoter, translated sequence(s) or non-translated RNA sequence(s), and other regulatory elements.

[0092] In some embodiments, the modified HSV-1 vector of the invention further comprises one or more additional nucleic acid sequences, which can be introduced in an intergenic region (IR) or a genic region. In embodiments, the additional nucleic acid sequence is introduced in an intergenic region (IR) different from the IR for the first and second transgenes. In some embodiments, the additional nucleic acid sequence is introduced in a genic region.

[0093] In some embodiments, the additional nucleic acid sequence comprises exogenous or endogenous DNA or combinations thereof into the modified HSV-1 vector of the invention. In embodiments, the additional nucleic acid sequence provides exogenous DNA into the modified HSV-1 vector. In embodiments, the additional nucleic acid sequence provides endogenous DNA into the modified HSV-1 vector. In embodiments, the additional nucleic acid sequence provides combinations of exogenous and endogenous DNA into the modified HSV-1 vector. In some embodiments, the introduction of one or more additional nucleic acid sequences brings the size of a modified HSV-1 vector produced from the HSV-1 pre-vector to a final size of about 152 kb. In other words, the size of the genome of the modified HSV-1 vector produced from the HSV-1 pre-vector is comparable to the size of the genome of the wild- type HSV-1 genome.

[0094] In some embodiments, the additional nucleic acid sequence to be introduced is selected from “stuffer” as defined herein, one or more HSV-1 essential genes, one or more HSV-1 non- essential genes, one or more exogenous genes of interest (as defined herein) or combinations thereof.

[0095] As used herein, the term, one or more exogenous genes of interest can include, but are not limited to, a reporter gene (e.g., GFP, RFP, luciferase, or fused protein, etc.) driven by an inducible, transient or persistent promoter serving as internal expression control or for biodistribution studies; recombinases driven by an inducible promoter to allow in vivo modifying cellular or viral genes; antibiotic resistance genes such as chloramphenicol; elements of the Tetracycline inducible system (TRE); any foreign DNA encoding a gene of interest, or combinations thereof. The use of reporter genes such as, but not limited to, firefly Luciferase, mCherry, mScarlet, RFP, GFP, CFP, or mGreen Lantern can facilitate the identification of the recombined genome, to assess the stability of the stuffer and to score both infectious particles (PFU) and transducing units (TU).

[0096] In some embodiments, the additional nucleic acid sequence is a DNA stuffer. In some embodiments, the additional nucleic acid sequence is one or more HSV-1 essential genes. In some embodiments, the additional nucleic acid sequence is a combination of stuffer and one or more HSV-1 essential genes.

[0097] In some embodiments, the additional nucleic acid sequence is one or more HSV-1 non- essential genes. In some embodiments, the additional nucleic acid sequence is a combination of stuffer and one or more HSV-1 non-essential genes. In some embodiments, the additional nucleic acid sequence is a combination of stuffer, one or more HSV-1 essential genes, and one or more HSV-1 non-essential genes. In some embodiments, the additional nucleic acid sequence is a combination of one or more HSV-1 essential genes and one or more HSV-1 non-essential genes. In some embodiments, the additional nucleic acid sequence is one or more exogenous genes of interest. In some embodiments, the additional nucleic acid sequence is a combination of stuffer and one or more exogenous genes of interest. In some embodiments, the additional nucleic acid sequence is a combination of stuffer, one or more HSV-1 essential genes, and one or more exogenous genes of interest. In some embodiments, the additional nucleic acid sequence is a combination of stuffer, one or more HSV-1 non-essential genes, and one or more exogenous genes of interest. In some embodiments, the additional nucleic acid sequence is a combination of one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, and one or more exogenous genes of interest. In some embodiments, the additional nucleic acid sequence is a combination of one or more HSV-1 essential genes and one or more exogenous genes of interest. In some embodiments, the additional nucleic acid sequence nucleic acid sequence is a combination of one or more HSV-1 non-essential genes and one or more exogenous genes of interest.

[0098] In any embodiments herein, the size of the stuffer will be determined based on the size of the other nucleic acid sequences introduced (e.g., one or more transgenes and / or one or more nucleic acids encoding cell targeting proteins) into the modified HSV-1 vector.

[0099] In some embodiments, the stuffer can be introduced into the modified HSV-1 vector of the invention as a single, long nucleic acid sequence. In some embodiments, the stuffer can be introduced into the modified HSV-1 vector of the invention as two or more stuff ers of the same or different lengths. Staffers can be inserted in the same DNA regions as the first or the second transgene. In preferred embodiments, the one or more staffers are inserted in different DNA regions from the first and second transgene.

[0100] In some embodiments, the stuffer can be introduced as several, smaller stuffer in different intergenic regions of the modified HSV-1 genome. In some embodiments, the smaller stuffer can be the same or different lengths.

[0101] It is important to note that a BAC sequence, when present in the HSV-1 pre-vector, is removed when preparing the modified HSV-1 vector of the invention. As such, the size of the BAC sequence is not considered when determining the size of the stuffer so that the size of the modified HSV-1 vector is about 152 kbp.

[0102] As used herein, the term “stuffer” refers to any random non-coding DNA sequence, noncoding RNA sequence, and / or one or more genes of interest, or combinations thereof. The primary purpose of the stuffer is to reintroduce a nucleic acid of an appropriate size (in base pairs) so that the size of the modified HSV-1 vector, based on the starting size of the Pre-HSV-1 vector, is at or close to the size of the wild type HSV-1 genome, i.e., about 152 kbp, after all other nucleic acids of interest (e.g., one or more transgenes and / or one or more exogenous nucleic acids and / or one or more endogenous nucleic acids) has been added to the Pre-HSV-1 vector. Considerations for the stuffer include the nature of the stuffer, the size of the stuffer, and the placement of the stuffer in the genome of the modified HSV-1 vector of the invention. Therefore, the design of the stuffer is determined last after all other design choices for the modified HSV-1 vector have been made. In embodiments, the stuffer is a DNA sequence. For example, in one embodiment a scrambled sequence from the HSV-1 genome (e.g., scrambled HSV-1 DNA) can be used. In some embodiments, the stuffer can be a scrambled nucleotide sequence of genes deleted from the HSV-1 genome.

[0103] In embodiments, the stuffer can originate from cellular DNA introns.

[0104] The production of any modified HSV-1 vector prepared from the HSV-1 pre-vector will thus require the concurrent construction of a cell line simultaneously complementing the deleted essential genes and optionally one or more of these non-essential genes.

[0105] Therefore, to facilitate growing, producing, and propagating the modified HSV-1 vectors of the invention and producing stocks thereof, an aspect of the invention provides a complementing cell line, which complements the genes deleted from the HSV genome. Thus, a preferred complementing cell according to the present invention is derived from a cell type that complements the HSV genes that have been deleted. Such cells can be engineered to express such genes by methods known in the art (e.g., by introducing expression cassettes within the cells so that they express the genes from genetic constructs other than the HSV genome, such as the cellular chromosomes).

[0106] Additionally, the complementing cell line can be engineered to express a gene encoding a selectable marker, such as markers typically employed in engineering packaging cells or cells expressing any other foreign gene. Suitable selectable genes include those conferring resistance to neomycin / G418, hygromycin, blasticidin, puromycin, zeocin, and the like.

[0107] It will be understood that methods for engineering a source cell type (e.g., Vero cells) to contain expression constructs encoding the deleted HSV proteins, as well as other proteins (such as the recombinase and / or the selectable gene product) are known to persons of ordinary skill. For example, the gene of interest with a selectable marker can be subcloned into lentiviral vectors, the source cell infected with the lentiviral vectors, selected for expression of the marker (e.g., blasticidin resistance), and then expression of the gene of interest confirmed.

[0108] The modified HSV-1 vector can be produced via any available cell lines. For example, the manufacturing of the modified HSV-1 vectors utilizes a complementing cell line. The inventive complementing cell comprising the modified HSV-1 vectors can be propagated and cloned. Thus, the invention provides a clonal population, i.e., a cell line, comprising or consisting of or essentially of the complementing cell line as described herein.

[0109] Using the inventive complementing cells of the invention, the inventive modified HSV-1 vector can be propagated. Accordingly, the invention provides a method of propagating the modified HSV-1 vector of the present invention. In accordance with the inventive method, the complementing cell line is infected with the modified HSV-1 vector and then cultured until plaques form. The viral population is amplified by repeated transfer of infectious particles to increasingly large, fresh populations of the complementing cells. For these repeated transfers, multiplicity of infection (MOI) can be between about 0.001 pfu / cell and about 0.03 pfu / cell. Ultimately, the inventive vectors (as packaged viruses) are purified from the cells at 90% cytopathic effect.

[0110] Generally, the inventive modified HSV-1 vector is most useful when enough of the virus can be delivered to a cell population to ensure that the cells are confronted with a suitable number of viruses. Thus, the present invention provides a stock, preferably a homogeneous stock, comprising the inventive modified HSV-1 vector. The preparation and analysis of HSV stocks is well known in the art. For example, a viral stock can be manufactured in roller bottles containing cells infected with the HSV-1 vector. The viral stock can then be purified on a continuous gradient, and aliquoted and stored until needed. Viral stocks vary considerably in titer, depending largely on viral genotype and the protocol and cell lines used to prepare them. Preferably, such a stock has a viral titer of about 106pfu / ml or even more preferably about 107pfu / ml (or at least about such values). In still more preferred embodiments, the titer can be about 108pfu / ml, or about 109pfu / ml (or at least about such values), and high titer stocks of about 1010pfu / ml or about 1011pfu / ml or even about 1012pfu / ml (or at least about such values) are most preferred. Thus, the titer of the HSV-1 vector stock according to the present invention can vary from about 106pfu / ml to about 1012pfu / ml (preferably between about 109to about 101 1pfu / ml). The invention additionally provides a composition comprising the modified HSV-1 vector of the invention and a physiologically-acceptable carrier. The carrier of the composition can be any suitable carrier for the vector. The carrier desirably is a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). Pharmaceutically acceptable carriers are well known and are readily available. The choice of carrier will be determined, at least in part, by the particular vector and the particular method used to administer the composition. The composition can further comprise any other suitable components, especially for enhancing the stability of the composition and / or its end-use. Accordingly, there is a wide variety of suitable formulations of the composition of the invention. The following formulations and methods are merely exemplary and are in no way limiting.

[0111] Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multidose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use.

[0112] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0113] In addition, the composition can comprise additional therapeutic or biologically- active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present. Factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the vector and physiological distress. Immune system suppressors can be administered with the composition method to reduce any immune response to the vector itself or associated with a disorder. Alternatively, immune enhancers can be included in the composition to up-regulate the body's natural defenses against disease. Antibiotics, i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders. The modified HSV-1 vector is capable of both transient expression of one or more first transgenes and persistent expression of one or more second transgenes (such as a therapeutic transgene). Using the modified HSV-1 vector (and stocks and compositions comprising the vector), the invention provides a method of expressing a transgene within a nucleated cell, especially a non-complementing cell. In accordance with the method, the inventive vector is exposed to the cell under conditions suitable for the vector to infect the cell. Once the cell is infected, the transgene will be transcribed (expressed) within the cell, provided the promoter within the transgene is one which is active in the cell and that the transgene is not suppressed by another regulatory mechanism (e.g., the microRNAs discussed herein). In other words, the inventive vectors serve as gene transfer and expression vectors within mammalian cells.

[0114] The invention can be employed to express transgene(s) within cells either in vivo or in vitro, as desired. For use in vivo, the cell can be any type of desired cell, such as exocrine secretory cells (e.g., glandular cells, such as salivary gland cells, mammary gland cells, sweat gland cells, digestive gland cells, etc.), hormone secreting gland cells (e.g., pituitary cells, thyroid cells, parathyroid cells, adrenal cells, etc.), ectoderm-derived cells (e.g., keratinizing epithelial cells (e.g., making up the skin and hair), wet stratified barrier epithelial cells (e.g., of the cornea, tongue, oral cavity, gastrointestinal tract, urethra, vagina, etc.), cells of the nervous system (e.g., peripheral and central neurons, glia, etc.), mesoderm- derived cells, cells of many internal organs (such as kidney, liver, pancreas, heart, lung) bone marrow cells, and cancerous cells either within tumors or otherwise. Preferred non-limiting examples of cells suitable for infection by the inventive vectors include liver cells, lung cells, epithelial cells, cardiac cells, adipose cells, muscle cells, stem cells, and cancer cells.

[0115] When used in vivo, the inventive method can treat a disease or a condition within a subject, when the transgene within the vector encodes one or more prophy lactically- or therapeutically-active proteins, polypeptides, or other factor (e.g., non-coding RNA (ncRNA) such as siRNA or miRNA). Thus, the invention provides a method of treating a disease or condition in a subject, comprising administering the vector of the present invention to the subject, in an amount and at a location sufficient to infect cells of the subject such that the transgene is expressed within the cells of the subject, and wherein the transgene encodes one or more prophylactically or therapeutically active proteins, polypeptides or ncRNA. For example, the disease or condition can be a type of cancer, in which the transgene can encode an agent that enhances tumor killing activity (such as TRAIL or tumor necrosis factor (TNF)). Additionally, the transgene can encode, but is not limited to, an agent suitable for the treatment of conditions such as muscular dystrophy (a suitable transgene encodes Dystrophin), cardiovascular disease (suitable transgenes include, e.g., SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (suitable transgenes include, e.g., NGF, BDNF, GDNF, NT- 3, etc.), chronic pain (suitable transgenes encode GlyRal , an enkephalin, or a glutamate decarboxylase (e.g., GAD65, GAD67, or another isoform), lung disease (e.g., CFTR), or hemophilia (suitable transgenes encode, e.g., Factor VIII or Factor IX).

[0116] In other embodiments, the inventive method can be used in vitro to cause expression of the transgene within cells in culture. Again, any type of cells can be infected in vitro with the inventive method, such as stem cells and fibroblasts, such as a human dermal fibroblast (HDF) or a human lung fibroblast (HLF). Other preferred types of cells for use in vitro include keratinocytes, peripheral blood mononuclear cells, hematopoietic stem cells (CD34+), or mesenchymal stem / progenitor cells. In one embodiment, the transgene(s) encode one or more factors that can affect the differentiation of the cell.

[0117] In embodiments, infecting a cell in vivo or in vitro with the vector, composition, or stock of the invention, the cell can be any mammalian nucleated cell for which it is desired to express the transgene. Thus, the vector can be employed to infect cells of many mammalian species. It is believed that the inventive methods can be applied in veterinary therapies for breeding, such as to express exogenous genes or supplement for deficient genes in animals. The animals for this invention can be any animal models that are permissive to HSV-1 infection, such as mice, rats, rabbits, guinea pigs, hamsters, or non-human primates such as macaques. Similarly, the inventive method can be employed in a veterinary context for companion animals.

[0118] The modified HSV-1 vectors of the invention can be used in vivo in humans as well, to provide for the expression of a prophylactically- or therapeutically -active agent, or factor, in a medical setting. The factor (supplied by expression of one or more of the transgenes within the inventive vectors) can be exogenous, or one that complements a genetic deficiency.

[0119] In some embodiments, the invention provides a kit comprising the modified HSV-1 vector according to the invention and instructions.

[0120] Features may be described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

[0121] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0122] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0123] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers with that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0124] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0125] As used herein, “heterogenous expression” refers to that multiple transgenes included in one vector, delivered to a cell at the same time, exhibit distinct expression kinetics. Namely, they have different expression periods. “Different expression periods” means the time lengths of the expression are different, regardless of whether the starting time for each expression period is the same or different from each other.

[0126] EXAMPLES

[0127] The following examples further illustrate the invention but should not be construed as limiting its scope. Example 1: To investigate the effect of the location of the transgenes on their kinetics of expression, nrHSV-1 vectors with the insertion of two transgenes at different locations are designed. The mGreenLantern reporter transgene has been inserted in the LAT region, allowing long-term expression in cells, while the mScarlet reporter transgene has been inserted in the long region between genes expressed only during the lytic cycle, hence providing a short-term expression. For example, insertion in the intergenic region between UL21 and UL22 (Figure 1), or UL26.5 and UL27 (Figure 2), or UL45-UL46 (Figure 3) are devised.

[0128] Example 2: In addition, as the location of the sites of heterochromatin nucleation in the HSV-1 genome are not fully understood, additional nrHSV-1 vectors are designed in order to evaluate the kinetic of mScarlet expression. While mGreenLantern is still located in the LAT region, mScarlet has been inserted in the unique short region between the lytic genes US1 and US2 (Figure 4), in the region deleted for the joint region (Figure 5), or in the long terminal repeat between the lytic genes ICP34.5 and ICPO (Figure 6).

[0129] Example 3: In addition, as the genic region and intergenic region may be differently repressed by the chromatin, additional nrHSV-1 vectors are designed to evaluate the kinetic of mScarlet expression. While mGreenLantern is still located in the LAT region, mScarlet has been inserted in lieu of the US1 gene, while preserving the US1 IE4 / 5 viral promoter (Figure 8), or in lieu of US12 gene, while preserving the US12 IE4 / 5 viral promoter (Figure 9), or in lieu of RL2 (ICPO) gene, while preserving the ICPO IE4 / 5 viral promoter (FigurelO).

[0130] In the following Examples, it was determined whether the above-described nrHSV-1 vectors allow the expression of multiple transgenes with distinct kinetics of expression, we perform in vitro and in vivo analysis of the expression of both reporter transgenes following infection with the nrHSV-1 vector in different cell types, as well as in mouse and rat models at several timepoints after infection.

[0131] To this end, HSV-bacterial artificial chromosomes (BAC) are generated by using “en passant” recombination and transfected in complementing African- green monkey kidney (Vero) cells to produce the nrHSV-1 particles described above.

[0132] Then, non-complementing glutamatergic neurons derived from human iPS cells and dopaminergic neurons derived from human iPS cells are infected at different MOI (0, or 0.5) with one of the HSV-1 vectors described above. At different point post-infection (3 days, 8 days, 17 days or 21 days), the expression of mScarlet, located outside of the LAT region, and mGreenLantern, from the LAT region, are assessed by live fluorescence imaging and quantified by quantitative RT-PCR (qRT-PCR).

[0133] In parallel, the nrHSV-1 vectors are injected in BALB / cByJ mice, in the footpad of the animals. At different time points post injection (3 days, 1 week, 2 weeks, 3 weeks, 1 month) animals are euthanized and the dorsal root ganglions (DRG) are collected. Expression levels of both reporter transgenes in those tissues are quantified by dRT-PCR and groups are compared between each other.

[0134] Example 4: A time-course analysis of in vitro transgenes expression in iPSC-derived cortical glutamatergic neuronal cells infected with the viral construct of Figure 3 (also referred to herein as “construct 3”) (expressing mGreenLantern under the control of the EFla promoter (green channel) and mScarlet under the Ubiquitin C promoter (red channel)) was performed. Cells infected with the viral construct of Figure 3 were observed at 3-, 8-, and 21 -days postinfection (dpi), with non-infected cells serving as controls. As shown in Figure 11 A, in the green channel (top row), cells show strong and uniform mLantem fluorescence up to 8 dpi, as indicated by multiple fluorescent cells marked with arrows and insets. At 21 dpi, the green signal intensity decreases but maintains the same cellular distribution and morphology, demonstrating a stable expression pattern despite the reduction in brightness. In contrast, the red channel (middle row) reveals a progressive loss of mScarlet fluorescence over time. At 3 dpi, a strong and diffuse signal of mScarlet is observed in infected cells while at 8 dpi, mScarlet shows punctate intracellular localization, with varying intensity across cells. At 21 dpi, red fluorescence is barely detectable. The brightfield and merged images (bottom row) confirm the presence and integrity of the cells across all timepoints, with fluorescent signals co-localized in morphologically intact cells. Non-infected control cells exhibit no detectable fluorescence in either channel, confirming the specificity of the observed signals. Figure 11B provides a quantitative analysis of reporter gene expression in infected cells. The expression levels of the mGreenLantern (black circle) and mScarlet (white square) reporter genes in infected cells at 3-, 8-, and 21- dpi were measured by qRT-PCR. Values were normalized to the amount of viral episomes in each sample and expressed as a percentage of mGreenLantern expression at 3 dpi, which is set as 100%. At 3 dpi, mGreenLantern shows maximal expression (100%), while mScarlet is expressed at approximately 75% relative to mGreenLantern, indicating a lower initial transcriptional output from the UL45-UL46 intergenic region compared to the LAT region. At 8 dpi, mGreenLantern maintains a high expression level (-85%), consistent with the stable and intense green fluorescence observed microscopically. In contrast, mScarlet expression drops sharply to around 15%, reflecting the early onset of transcriptional silencing and matching the observed heterogeneity and reduction in red fluorescence. At 21 dpi, mGreenLantern further decreases to -40%, whereas mScarlet expression remains closed to 15%. These data confirm that, when inserted into the LAT region, the mGreenLantern is more stably expressed over time, while mScarlet, located in the intergenic UL45-UL46 region, is rapidly and progressively silenced, highlighting different regulatory control depending on the insertion site.

[0135] Example 5: A time-course analysis of in vitro transgenes expression in iPSC-derived cortical glutamatergic neurons infected with the viral construct of Figure 4 (also referred to herein as “construct 4”)(which encodes mGreenLantern and mScarlet reporters inserted into the LAT and US1-US2 InterGenic Regions, respectively) was performed. As shown in Figure 12A, live-cell fluorescence microscopy reveals robust and widespread mGreenLantern expression in infected iPSC-derived glutamatergic neurons at both 3 and 8 dpi, as shown by numerous intensely fluorescent green cells (arrows and insets, right and middle top panels). By 21 dpi, green fluorescence decreases in intensity but retains a consistent intracellular localization pattern, comparable to observations with the vector of Figure 3. In contrast, mScarlet fluorescence (middle row) shows a rapid decline. At 3 dpi, mScarlet is strongly expressed with diffuse red fluorescence throughout the cytoplasm. By 8 dpi, mScarlet fluorescence becomes heterogeneous among infected cells, with some cells retaining a diffuse cytoplasmic signal, while others display a punctate intracellular pattern, as highlighted by the variability in red fluorescence intensity and distribution across the population (middle row, middle panel). At 21 dpi, red signal is barely detectable, marking a near-complete repression of transgene expression from the US1-US2 InterGenic Region (middle raw, left panel). Brightfield and merged images confirm preservation of cell morphology and co-expression of the fluorescent signals in intact neurons (bottom raw). Non-infected control cells show no background fluorescence (left panels). Figure 12B shows that quantitative qRT-PCR analysis supports the microscopy findings. mGreenLantern (black circle) expression, normalized to viral episome content and set at 100% for 3 dpi, remains high at -100% at 8 dpi and declines to -55% at 21 dpi, indicating moderately decreasing but sustained expression from the LAT locus. In contrast, mScarlet (white square) expression is initially -80% at 3 dpi but drops to -40% at 8 dpi, a higher expression level than observed with the vector of Figure 3 at the same time point (-15%). At 21dpi, mScarlet level of expression drops to -30%. These transcriptional trends are consistent with the presence of a mixed population of cells: some maintaining high expression (diffuse signal), and others showing signs of transcriptional silencing or compartmentalization (punctate signal). Statistical significance at each time point was assessed using the Mann-Whitney test, with p-values indicated as follows: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

[0136] Example 6: The Expression levels of the two reporter genes (mGreenLantern and mScarlet) in iPSC-derived dopaminergic neurons infected with the nrHSV-1 vector of Figure 3 were determined. Two biological samples are shown per condition. The expression levels were normalized to viral episome copy number and expressed as a percentage relative to mGreenLantern expression at 3 dpi, which is set to 100%. Figure 13 provides qRT-PCR data quantifying mGreen Lantern and mScarlet reporter genes expression at 3 and 17dpi. At 3 dpi, mGreenLantern, inserted into the LAT region, exhibits robust expression (100%), while mScarlet, inserted into the UL45-UL46 intergenic region, shows even higher relative expression at 131%, suggesting a strong early transcriptional output from both loci. However, by 17 dpi, divergent expression trends emerge: mGreenLantern expression declines moderately to 59%, suggesting partial but sustained transcription from the LAT region. mScarlet expression drops sharply to 18%, reflecting substantial transcriptional silencing from the UL45-UL46 region. This trend highlights the differential transcriptional stability conferred by the two genomic contexts: while the LAT region maintains moderate transgene expression over time, the UL45-UL46 intergenic region is subject to rapid and persistent transcriptional repression.

[0137] Example 7: A comparison of the expression in dorsal root ganglia (DRGs) of the two fluorescent reporter transgenes (mGreenLantern (gray circles) and mScarlet (black squares)), for both constructs, of Figure 3 (Figure 14A) and of Figure 4 (Figure 14B) was performed in Balb / c mice over a 28-day period following injection. Vectors (1.8E+08PFU / animal) were injected in the footpad of female Balb / c mice of 7weeks old (8 animals per group). After footpad injection, nrHSV-1 vectors can efficiently transduce Dorsal Root Ganglia (DRG) neurons through retrograde transport from sciatic nerve endings, the primary innervation of the footpad, to neurons cells bodies mostly located in L4 to L6 DRGs. DRGs were collected at indicated time point (i.e. 3-, 7-, 14-, 21- and 28-dpi), and transgene expression in DRGs L4 to L6 is measured using qRT-PCR following nucleic acid extraction. Figure 14 depicts the percentage of positive animals for transgene expression following injection. Figure 14 A shows that the percentage of animals exhibiting detectable mGreenLantern transgene expression by qRT-PCR increases over time following injection with the vector construct of Figure 3. Specifically, 7 out of 8 animals had measurable levels of mGreenLantern transcript in the L4-L6 DRGs at 3 days post-injection (3 dpi), corresponding to approximately 85% positivity. This proportion rises to 100% from day 7 through day 28, indicating that mGreenLantern expression from the LAT region is not only robust but also stably maintained over time. In this context, an animal is considered positive when transgene expression is detectable above the assay threshold by qRT-PCR in the harvested DRG tissue. In contrast, the percentage of mScarlet-positive animals declines sharply after injection. At day 3, approximately 25% of the animals show detectable mScarlet expression, which further drops to around 12% by day 7, and becomes undetectable by day 14. This rapid loss of detectable signal indicates a transient and weak expression of the mScarlet transgene in animals injected with the vector of Figure 3. Figure 14B shows the percentage of animals with detectable transgene expression in the L4-L6 DRGs following injection with the vector construct of Figure 4, as measured by qRT-PCR. All animals (8 out of 8) exhibited detectable mGreenLantern expression at 3 days post- injection (3 dpi), and this 100% positivity was maintained consistently through days 7, 14, and 28, demonstrating strong and persistent transgene expression from the LAT region over time. As previously, an animal is defined as positive when the qRT-PCR signal for the transgene exceeds the assay detection threshold in at least one of the analyzed DRGs. In contrast, mScarlet expression in animals injected with the vector of Figure 4 decline rapidly following injection. While 90% of animals display detectable expression at day 3, this proportion drops to approximately 60% by day 7, about 12% by day 14, and is completely undetectable (0%) by day 21.

[0138] Example 8: The kinetics and magnitude of mGreenLantern and mScarlet transgene expression over time in DRGs from Balb / c mice following footpad injection of HSV-l-derived vectors of Figure 3 or of Figure 4 (as described in Example 7) were determined. Expression levels were measured by quantitative RT-PCR in DRGs L4 to L6 at 3-, 7-, 14-, 21-, and 28-dpi, and are presented relative to the level of mGreenLantern expression at day 3 for each condition. Statistical significance at each time point was assessed using the Mann-Whitney test, with p- values indicated as follows: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. Figures 15 A, B, and C correspond to animals injected with the vector of Figure 3, in which mGreenLantern is inserted into the LAT locus and mScarlet into the UL45-UL46 intergenic region. Figure 15A shows the expression kinetics of both reporters. mGreenLantern expression (black circle) is detectable at 3 dpi in the majority of animals and increases steadily over time, reaching approximately 2000% of its initial value by 14 dpi and stabilizing to a plateau up to 28 dpi. In contrast, mScarlet (grey square) expression is minimal across the time course, becoming nearly undetectable in all tested animals from 14 dpi onwards. Figure 15B shows a box-and- whisker plot of mGreenLantern expression levels over time for animals injected with the vector of Figure 3, illustrating a progressive increase in median values from day 3 to day 28. Figure 15C displays the expression of mScarlet normalized to the level of mGreenLantern at day 3. mScarlet reaches approximately 16% of the mGreenLantern reference at early time points (3 dpi) but falls to negligible levels by 7- to 14-dpi. Figures 15 D, E, and F present data from animals injected with the vector of Figure 4 in which mGreenLantern is inserted into the LAT locus and mScarlet into the US1-US2 intergenic region. Figure 15D indicates that mGreenLantern expression (black circle) is significantly higher than mScarlet at all time points, reaches a maximum of approximately 450% by day 14, relative to day 3. In contrast, mScarlet (grey square) expression is initially low and decreases rapidly, becoming nearly undetectable from 21 dpi onwards in all tested animals. Figure 15E illustrates mGreenLantern expression kinetic over time in animals injected with the vector of Figure 4, showing increase from day 3 to day 14 and then plateau up to 28dpi. Figure 15F shows mScarlet expression as a percentage of the mGreenLantern day 3 baseline. A small transient expression is observed at 3 dpi in 7 out of 8 animals, mScarlet levels decline rapidly at day 7 and remain close to background from 14 dpi onward. Notably, mScarlet expression levels in animals injected with vector of Figure 4 are slightly higher and persist slightly longer than in animals injected with vector of Figure 3, suggesting that positioning mScarlet in the US1-US2 intergenic region may delay, but not prevent, the onset of transcriptional silencing.

[0139] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0140] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. It will also be understood that none of the embodiments described herein are mutually exclusive and may be combined in various ways without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS1. A modified HSV-1 vector comprising at least two transgenes inserted within different genomic regions of the modified HSV-1 vector to provide distinct kinetic profiles of expression, wherein at least a first transgene is expressed for a first period and at least a second transgene is expressed for a second period after entering a cell, and wherein the first period is a shorter period than the second period.

2. The modified HSV-1 vector of claim 1, wherein the first transgene has a transient expression profile during the first period and the second transgene has a long-term expression profile during the second period.

3. The modified HSV-1 vector of claim 1 or 2, wherein the first period is no greater than about four weeks.

4. The modified HSV-1 vector of any one of claims 1-3, wherein the second period is at least about four weeks5. The modified HSV-1 vector of any one of claims 1-4, wherein the modified HSV-1 vector is a non-replicative HSV-1 (nrHSV-1) vector.

6. The modified HSV-1 vector of any one of claims 1-5, wherein the second transgene is a part of an expression cassette, wherein the expression cassette further comprises a promoter and optionally an enhancer.

7. The modified HSV-1 vector of any one of claims 1-6, wherein the first transgene is a part of an expression cassette, wherein the expression cassette further comprises a promoter and optionally an enhancer.

8. The modified HSV-1 vector of any one of claims 1-7, wherein the first transgene is only expressed before heterochromatinization after entering the cell.

9. The modified HSV-1 vector of any one of claims 1-8, wherein the second transgene is operably linked to at least one sequence conferring long-term expression.

10. The modified HSV-1 vector of any one of claims 1-9, the second transgene is introduced downstream of a chromatin insulator.

11. The modified HSV-1 vector of any one of claims 1-10, the second transgene is introduced between two chromatin insulators.

12. The modified HSV-1 vector of any one of claims 1-11, wherein the second transgene is introduced to a Latency Associated Transcripts (LAT) region or the ICP4 loci of the modified HSV-1 vector.

13. The modified HSV-1 vector of any one of claims 1-12, the first transgene is introduced into the intergenic region between two lytic genes.

14. The modified HSV-1 vector of claim 13, the two lytic genes are selected from the pairs of UL3-UL4, UL7-UL8, UL10.5-UL11, UL15-UL16, UL21-UL22, UL26.5-UL27, UL30-U131, U135-UL36, UL40-UL41, UL45-UL46, UL50-UL51, or UL55-UL56. US1-US2, and US9-US10.

15. The modified HSV-1 vector of claim 13, wherein the two lytic genes are ICP34.5 (i.e., y34.5) and ICPO.

16. The modified HSV-1 vector of any one of claims 1-12, the first transgene is introduced into a genic region expressed during the lytic cycle.

17. The modified HSV-1 vector of any one of claims 1-16, wherein the modified HSV-1 vector further optionally comprises one or more additional nucleic acid sequences.

18. The modified HSV-1 vector of any one of claims 1-17, wherein the modified HSV-1 vector further optionally comprises one or more st ffers.

19. The modified HSV-1 vector of any one of claims 1-18, wherein the size of the modified HSV-1 genome is about 143 kbp to about 154.5 kbp.

20. A method of delivering at least two transgenes into a cell for heterogeneous expression, wherein the method comprises contacting the cell with the modified HSV-1 vector according to any one of claims 1-19.

21. The method of claim 20, wherein the cell is a neuron, an epithelial cell, a muscle cell, a connective tissue cell, or a platelet.

22. A method of treating a disease in a subject in need, wherein the method comprises administering the modified HSV-1 vector according to any one of claims 1-19 to the subject.

23. The method of claim 22, wherein the first period ranges from about two hours to about three weeks, and wherein the second period ranges from about four weeks to about a lifelong time of the subject.

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