Multifunctional HSV-1 precursor vector

Abstract

The present invention relates to a viral expression backbone, viral expression vectors produced therefrom, and pharmaceutical compositions thereof, which are suitable for therapeutic or in vitro applications, as well as methods related thereto. In other words, the genome size of the modified HSV-1 vector generated by the HSV-1 precursor vector provided by the invention is equivalent to that of a wild type HSV-1 genome.

Description

Background Technology

[0001] Herpes simplex virus (HSV) is a complex, non-integrating DNA virus capable of infecting various human and animal cells. HSV comprises two serotypes: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). The HSV-1 genome is approximately 152 kilobase pairs (kbp) in size. It contains approximately 90 protein-coding genes and more than 12 microRNAs. The HSV-1 genome consists of two distinct segments, UL and US, each flanked by inverted repeat sequences that encode two copies of key genes.

[0002] HSV-1-based vectors have been investigated for use as gene transfer vectors, including potential therapeutic applications in human patients. These vectors are engineered to contain transgenes that can be expressed in cells or tissues. For non-replicating recombinant vectors, the vector genome is further modified to remove essential immediate early (IE) genes, such as ICP27 and ICP4, making the virus completely defective and unable to express early (E) genes involved in viral genome replication and late (L) genes necessary for progeny viral particle assembly. These replication-deficient viruses can be cultured on complementary cells expressing (complementary) products of the missing genes and then used to infect non-complementary cells, where the viral genome expresses only the therapeutic transgenes. However, removing genes from the HSV genome can negatively impact vector productivity, genome stability, and / or transgene expression.

[0003] Despite some significant progress in this field, obtaining large quantities of high-titer, high-purity, and non-pathogenic viral vectors remains difficult and costly.

[0004] Therefore, there is still a need to develop non-replicating HSV-1 vectors that can be modified to contain more than one target transgene, are non-toxic to cells or tissues, and can be efficiently amplified while maintaining high transgene expression capacity. Summary of the Invention

[0005] This invention relates to a viral expression backbone, a viral expression vector derived from the backbone, and pharmaceutical compositions thereof, which are suitable for therapeutic or in vitro applications, and also to methods thereof.

[0006] In one embodiment, the present invention provides an HSV-1 pre-vector containing an HSV-1 genome, wherein non-essential genes, essential genes, or combinations thereof have been deleted to obtain a genome of less than 130 kbp and greater than 75 kbp.

[0007] In one embodiment, the present invention provides a modified HSV-1 vector comprising an HSV-1 precursor vector according to the invention, wherein one or more nucleic acids and one or more stuffers (as defined herein) are introduced into the HSV-1 precursor vector. In some embodiments, the one or more nucleic acids are target transgenes. In some embodiments, the transgenes are part of an expression cassette.

[0008] In one embodiment, the present invention provides a kit comprising an HSV-1 precursor vector according to the invention and instructions for use. In some embodiments, the kit may also optionally comprise a plurality of first vials, each first vial independently containing a filler fragment of a different length. In some embodiments, the kit may also optionally comprise a plurality of second vials, each second vial independently containing nucleic acid encoding a cell-targeting protein. Attached Figure Description

[0009] Figures 1A to 1J Various embodiments of the HSV-1 precursor carrier according to the present invention are shown.

[0010] Figure 1A The skeleton of the universal BAC-A_1del single-copy vector is shown.

[0011] Figure 1B : Universal BAC-A dual-copy vector backbone.

[0012] Figure 1C : General BAC-B1 △-gD redirection carrier skeleton.

[0013] Figure 1D : General △-ICP0 redirection carrier skeleton.

[0014] Figure 1E : General △-gD + △-ICP0 redirection carrier skeleton.

[0015] Figure 1F : Universal single-copy BAC-A_1del + △UL39-41 vector skeleton.

[0016] Figure 1G : Universal single-copy BAC-A_1del + △UL39-41 + △UL43-47 vector skeleton.

[0017] Figure 1H : Universal skeleton single copy BAC-A_1del + △UL39-41 + △UL43-47 + △US2-US6 carrier skeleton.

[0018] Figure 1I: Universal skeleton single copy BAC-A_1del + △UL39-41 + △UL43-47 + △US7-US12 vector skeleton.

[0019] Figure 1J : Universal skeleton single copy BAC-A_1del + △UL39-41 + △UL43-47 + △US2-US12 carrier skeleton.

[0020] Figures 2A to 2F Various embodiments of inserting filler fragments into an HSV-1 precursor carrier according to the present invention are shown.

[0021] Figure 2A : Universal BAC-A_1del single-copy vector skeleton, which contains filling fragments in △-ICP4.

[0022] Figure 2B The general BAC-A_1del single-copy vector backbone contains filler fragments in the Δ-ICP4 and intergenic regions.

[0023] Figure 2C : General BAC-A_1del single-copy vector skeleton, which contains filling fragments in △-joint.

[0024] Figure 2D The universal single-copy vector backbone is BAC-A_1del + △UL39-41 + △UL43-47 + △US2-US12, which contains filler fragments in the intergenic regions and △-joint 1 and △-joint 4.

[0025] Figure 2E : General BAC-B1 △-gD redirection carrier skeleton.

[0026] Figure 2F The universal BAC-A_filler fragment is located in the linker region, and the transgene is located in the LAT region.

[0027] Figure 3 An HSV-1 precursor vector is shown, which is a modified HSV-1 vector backbone that has optimal size after transfection of mammalian cells and simultaneous excision of the BAC sequence. Figure 3 The HSV-1 precursor vector without the filler fragment is shown, with a size of approximately 140,978 bp (including the BAC sequence) and approximately 131,533 bp without the BAC sequence.

[0028] Figures 4A to 4C Various implementation schemes of the modified HSV-1 vector are shown.

[0029] Figure 4AAn embodiment of the modified HSV-1 vector is shown, which contains the target nucleic acid sequence (i.e., the transgene) and does not contain filler fragments. After the BAC sequence is removed, the final DNA size is approximately 131,559 bp.

[0030] Figure 4B An embodiment of a modified HSV-1 vector is shown, which contains a target nucleic acid sequence (i.e., a transgene) and a filler fragment. After transfection into mammalian cells, the BAC module is excised, and the size is approximately 162,978 bp.

[0031] Figure 4C It shows Figure 4B The modified HSV-1 vector in the BAC module was approximately 153,533 bp in size after being automatically removed.

[0032] Figures 5A to 5C The image shows the results of sequencing a modified HSV-1 vector containing a filler fragment using Oxford Nanopore Technology.

[0033] Figure 5A : Figure 4C The DNA sequencing results of the modified HSV-1 vector shown (excluding filler fragments).

[0034] Figure 5B : Figure 4A The DNA sequencing results of the modified HSV-1 vector are shown (showing amplicon population).

[0035] Figure 5C DNA sequencing results of the modified HSV-1 vector, with an original size of 138,012 bp (showing DNA region repetition (9,000 bp)). Recombinant byproduct amplicon (due to the short genome size) is also present. Figure 5B ) and genome duplication ( Figure 5C (This is marked with a red arrow.)

[0036] Figure 6 This study illustrates the impact on the genomic stability of various modified HSV-1 vectors of different sizes during multiple passages. Detailed Implementation

[0037] To provide a clear and concise description of the invention, various features may be described herein, which may be part of the same or different aspects or embodiments of the invention. Those skilled in the art will understand that the scope of the invention may include embodiments having all or some of the features described herein, which may be part of the same or different embodiments.

[0038] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein include both singular and plural references.

[0039] The numerical range is described in terms of endpoints, which includes all numbers and fractions within the corresponding range, as well as the endpoints.

[0040] In this disclosure, various aspects of the invention may be described using a scope format. It should be understood that the use of a scope format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the invention. Therefore, a description of a scope should be considered as explicitly disclosing all possible sub-scopes and the specific values ​​within that scope. For example, a description of a scope such as "1 to 6" should be considered as explicitly disclosing sub-scopes such as "1 to 3", "1 to 4", "1 to 5", "2 to 4", "2 to 6", "3 to 6", etc., and the specific values ​​within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This principle applies regardless of the size of the scope.

[0041] The term “about” as used herein is to be understood by one of ordinary skill in the art and will vary slightly depending on the context in which it is used. In this document, when referring to measurable values ​​such as quantity or duration, the term “about” is intended to cover deviations from a specified value of ±20% or ±10%, including deviations of ±5%, ±1%, and ±0.1%, provided that such deviations are appropriate for performing the disclosed methods.

[0042] The wild-type HSV-1 genome is approximately 152 kbp in size, containing about 90 protein-coding genes and at least 12 microRNAs. However, the majority of this genome encodes non-essential genes, which can, in principle, be deleted individually without significantly interfering with viral replication and packaging in cultured cells. Furthermore, more than one essential gene can be deleted, but in this case, the virus can only replicate in the presence of a complementary system (e.g., a complementary cell line) that provides proteins not expressed by the vector genome. Therefore, the HSV-1 genome can be engineered to delete non-essential genes, essential genes, or combinations thereof. In summary, these deletions can create vast genomic space, allowing for the introduction and delivery of very large amounts of exogenous DNA.

[0043] HSV-1 precursor vector

[0044] In one embodiment, the present invention provides a miniature HSV-1 backbone (also referred to as "miniature HSV-1" or "HSV-1 precursor vector") wherein non-essential genes, essential genes, or combinations thereof have been deleted from the HSV-1 genome, thereby obtaining a genome backbone of less than 130 kb and greater than 75 kb. The HSV-1 precursor vector described in this invention refers to a "backbone" in which more than one nucleic acid may be inserted, with or without external control elements.

[0045] According to the present invention, the qualifier "essential" in "essential gene" or "non-essential gene" means that a given gene is essential (or non-essential) for the replication and packaging of the viral genome (thereby producing infectious progeny viral particles). Essential genes for HSV-1 include UL1, UL5-UL9, UL12, UL14, UL15, UL17-UL19, UL22, UL25-UL38, UL42, UL48, UL49, UL52, UL54, US6, and ICP4 (2 copies). HSV-1 non-essential genes include ICP34.5 (2 copies), ICP0 (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, and US7-US12.

[0046] In one implementation, the removable gene clusters include, but are not limited to: genes UL2, UL3, UL4 (10.200-12.600); genes UL10, UL11 (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); and genes UL50, UL51 (107.700-109.100). One copy of genes UL55 and UL56 (115,400-117,100); one copy of genes LAT, ICP0, and 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 a second copy of gene ICP0 (when the first copy in the LAT, ICP0, and UL34.5 gene cluster has been removed).

[0047] In some embodiments, the HSV-1 precursor vector contains at least 23 kb of deleted genome. In some embodiments, the HSV-1 precursor vector contains at least 30 kb of deleted genome. In some embodiments, the HSV-1 precursor vector contains at least 40 kb of deleted genome. In one embodiment, the HSV-1 precursor vector contains at least 45 kb of deleted genome. In some embodiments, the HSV-1 precursor vector contains at least 50 kb of deleted genome. In some embodiments, the HSV-1 precursor vector contains at least 55 kb of deleted genome. In some embodiments, the HSV-1 precursor vector contains at least 60 kb of deleted genome. In some embodiments, the HSV-1 precursor vector contains at least 65 kb of deleted genome. In some embodiments, the HSV-1 precursor vector contains at least 75 kb of deleted genome.

[0048] In one embodiment, the HSV-1 precursor vector contains 25 kbp to 80 kbp of deleted genome. In some embodiments, the HSV-1 precursor vector contains 30 kbp to 75 kbp of deleted genome. In some embodiments, the HSV-1 precursor vector contains 35 kbp to 70 kbp of deleted genome. In some embodiments, the HSV-1 precursor vector contains 40 kbp to 60 kbp of deleted genome.

[0049] In some embodiments, the present invention provides an HSV-1 precursor vector containing the HSV-1 genome, wherein non-essential genes, essential genes, or combinations thereof have been deleted to obtain a genomic backbone of less than 130 kbp and greater than 75 kbp.

[0050] In some implementations, the HSV-1 precursor vector also contains a bacterial artificial chromosome (BAC) sequence. In this document, a BAC is a genetically engineered DNA molecule used in a manner that allows for amplification in bacteria as a circular artificial chromosome. The BAC vector is a plasmid constructed using the E. coli F factor origin of replication, thus maintaining a single copy in each cell. These vectors are capable of holding DNA fragments up to 300 kb in length (see [link to implementation details]). Figure 3 (and Figure 4, etc.).

[0051] While it is theoretically possible to delete all non-essential HSV-1 genes from the BAC amplified in bacteria, simultaneously deleting all of these genes would likely render the virus inactive, reducing its virulence to the point where it cannot be effectively cultured in mammalian cells. Furthermore, the size of the HSV-1 precursor vector genome itself is also important, as genomes that are too short or too long cannot be properly packaged, thus requiring size compensation.

[0052] Importantly, the HSV-1 precursor vector of this invention must retain sufficient HSV-1 genome to avoid becoming an HSV-1 amplicon. "Amplicon or amplicon vector" refers to a helper-dependent vector whose genome lacks most or all of the HSV genes encoding viral proteins. The genome of an amplicon vector is tandem DNA composed of multiple tandem plasmids (called amplicon plasmids) carrying a DNA replication origin and a packaging signal from the HSV-1 genome. In cells expressing the full structure, replication, and DNA packaging functions of HSV-1, the amplicon plasmids are amplified into long, end-to-end tandem structures via rolling circle replication due to the presence of the HSV-1 genome as a helper factor. These tandem structures are then cleaved and packaged into HSV-1 viral particles, each viral particle capable of encapsulating up to one genome size (Kwong and Frenkel, 1985; Bataile and Epstein, 1997). Therefore, an amplicon vector is tandem plasmid DNA encapsulated within an HSV-1 particle.

[0053] Modified HSV-1 vector

[0054] As described herein, the HSV-1 precursor vectors of the present invention contain deletions to the HSV genome, such that the genomic backbone length of the HSV-1 precursor vector is less than 130 kbp and greater than 75 kbp. As mentioned above, these precursor vector "backbone" portions of the present invention serve as vector templates, and the deleted portions create genomic space, allowing the introduction of more than one target nucleic acid sequence. Introducing more than one target nucleic acid sequence (e.g., a target transgene) increases the genome size (i.e., base pair number) of the modified HSV-1 vector obtained by the present invention. While not wishing to be bound by any particular theory, it is believed that when the size of the modified HSV-1 vector of the present invention is similar to that of the wild-type HSV-1 genome (152 kbp), the resulting modified HSV-1 vector exhibits higher genomic stability, thereby enhancing vector activity.

[0055] In some embodiments, the present invention provides a modified HSV-1 vector comprising one or more target nucleic acid sequences and one or more filler fragments, wherein the size of the modified HSV-1 vector genome is approximately 138 kbp to approximately 168 kbp.

[0056] In some embodiments, the present invention provides a modified HSV-1 vector comprising one or more target nucleic acid sequences and one or more filler fragments, wherein the size of the modified HSV-1 vector genome is approximately 142 kbp to approximately 160 kbp.

[0057] In any of the embodiments disclosed herein, at least one of the more than one target nucleic acid sequences encodes a target protein.

[0058] In any of the embodiments disclosed herein, the more than one target nucleic acid sequence comprises more than one target transgene, more than one nucleic acid encoding a cell-targeting protein, or a combination thereof. In any of the embodiments disclosed herein, the more than one nucleic acid sequence comprises more than one target transgene. For clarity, the term "more than one target nucleic acid sequence" does not include the more than one filler fragment described herein.

[0059] In any of the embodiments disclosed herein, more than one target nucleic acid sequence may be introduced into the HSV-1 precursor vector backbone using site-specific recombination (SSR), homologous recombination (HR), or bridging mutation techniques. In any of the embodiments disclosed herein, more than one target nucleic acid sequence may be introduced using homologous recombination or bridging mutation techniques.

[0060] In some implementations, the modified HSV-1 vector contains one or more transgenes and one or more filler fragments, wherein the modified HSV-1 vector is capable of continuously expressing one or more transgenes, such as therapeutic transgenes.

[0061] In any embodiment described herein, after adding one or more filler fragments and one or more target nucleic acids, the size of the modified HSV-1 genome is between approximately 138 kbp and 168 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 142 kbp and 164 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 142 kbp and 155 kbp. In a preferred embodiment described herein, the size of the modified HSV-1 genome is between approximately 142 kbp and 153 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 147 kbp and 159 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 147 kbp and 156 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 150 kbp and 156 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is approximately 153 kbp.

[0062] In any embodiment described herein, after adding one or more filler fragments and one or more target nucleic acids, the size of the modified HSV-1 genome is between approximately 142 kbp and 160 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 142 kbp and 158 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 142 kbp and 155 kbp. In a preferred embodiment described herein, the size of the modified HSV-1 genome is between approximately 142 kbp and 153 kbp.

[0063] In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 143 kbp and 160 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 143 kbp and 158 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 143 kbp and 155 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 143 kbp and 153 kbp.

[0064] In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 145 kbp and 160 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 145 kbp and 158 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 145 kbp and 155 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 145 kbp and 153 kbp.

[0065] In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 147 kbp and 160 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 147 kbp and 158 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 147 kbp and 155 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 147 kbp and 153 kbp.

[0066] In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 149 kbp and 160 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 149 kbp and 158 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 149 kbp and 155 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 149 kbp and 153 kbp.

[0067] In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 150 kbp and 155 kbp. In any embodiment described herein, the size of the modified HSV-1 genome is between approximately 150 kbp and 153 kbp.

[0068] As used herein, the term "filler fragment" refers to any random non-coding DNA sequence and / or one or more target genes, or a combination thereof. The primary purpose of the filler fragment is to reintroduce appropriately sized nucleic acids (in base pairs) such that, after all other target nucleic acids (e.g., one or more transgenic and / or one or more exogenous and / or one or more endogenous nucleic acids) are added to the HSV-1 precursor vector, the size of the modified HSV-1 vector (based on the initial size of the HSV-1 precursor vector) reaches or approaches the size of the wild-type HSV-1 genome, approximately 152 kbp. Factors considered in the selection of the filler fragment include its nature, size, and location within the modified HSV-1 vector genome of this invention.

[0069] Therefore, the design of the filler fragment is performed last, after all other design options for the modified HSV-1 vector have been determined. For example, if the HSV-1 precursor vector of the present invention contains 105 kilobits (kbps) and a transgene containing 5 kbps is introduced (as described herein), then one or more filler fragments with a total length of about 33 kbps to about 58 kbps should be introduced into the modified HSV-1 vector. Alternatively, in this case, one or more filler fragments with a total length of about 36 kbps to about 50 kbps may also be introduced. Alternatively, in this case, one or more filler fragments with a total length of about 39 kbps to about 47 kbps may also be introduced. Alternatively, in this case, one or more filler fragments with a total length of about 42 kbps may also be introduced.

[0070] In some embodiments, the one or more filler fragments comprise non-coding DNA sequences. For example, in one embodiment, a scrambled sequence from the HSV-1 genome (e.g., scrambled HSV-1 DNA) can be used. The scrambled sequence of HSV-1 DNA may contain a sequence of no more than 20 nucleotides, but each can represent the entire genome to maintain a similar GC content and “natural style” (e.g., “natural behavior”) to HSV-1 DNA.

[0071] In some embodiments, the GC content of the one or more filler fragments is 60% to 75%. In some embodiments, the GC content of the filler fragments is 65% to 69%. In a preferred embodiment, the GC content of the filler fragments is 67% to 68%.

[0072] Sequences of no more than 20 nucleotides can be repeated until one or more filler fragments reach the desired length. These sequences can be derived from non-coding DNA to avoid introducing transcriptionally significant signals. Furthermore, sequences such as Kozak sequences, start codons, repetitive sequences, potential miRNA clusters, and long palindromic sequences should be avoided. Additionally, sequences present in the human genome or production cell line genomes should not be included to avoid vector genome recombination. Stop codons can be routinely introduced to avoid long open reading frames. In general, the filler fragments of random DNA should avoid any biological significance, limiting their use to increasing size.

[0073] In some implementations, one or more filler fragments may be out-of-order nucleotide sequences of genes deleted from the HSV-1 genome.

[0074] In some implementations, one or more filler fragments may be derived from cellular DNA introns that have been modified to avoid vector genome recombination.

[0075] In some implementations, the filler fragments may be divided into two or more fragments and inserted into different locations in the HSV-1 precursor vector genome. In a preferred embodiment, the two or more filler fragments should not have homologous sequences to avoid internal recombination within the vector genome.

[0076] In some implementations, the primary function of one or more filler fragments can be achieved using any random non-coding DNA sequence and / or one or more target genes, or combinations thereof. That is, the "filler fragment" can restore the size of the modified HSV-1 vector genome to approximately 152 kb by using any random non-coding DNA sequence alone. Alternatively, in some implementations, the function of the filler fragment can be achieved by using one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, one or more target exogenous genes (as defined herein), or combinations thereof (with or without using one or more random non-coding DNA sequences).

[0077] The term “transgenic” refers to a specific coding nucleic acid sequence that encodes or does not encode RNA and / or polypeptides or polypeptide fragments, which will be expressed in cells in which the nucleic acid sequence is introduced. “Transgenic” includes: (1) a nucleic acid sequence that is not naturally present in the cell (i.e., a heterologous nucleic acid sequence); (2) a mutant form of a nucleic acid sequence, i.e., a mutant of a naturally present nucleic acid sequence introduced into the cell; (3) a nucleic acid sequence used to add an additional copy of the same (i.e., homologous) or similar natural nucleic acid sequence to the introduced cell; or (4) a silent natural or homologous nucleic acid sequence that is induced to be expressed in the introduced cell. “Mutant” refers to a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or natural sequence, i.e., a mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions and / or insertions. In some cases, a transgenic may contain a sequence encoding a leader peptide or signal sequence to secrete the transgenic product out of the cell; or a transgenic may contain both a leader peptide or signal sequence and a membrane anchoring peptide; or it may even be a fusion protein of two natural proteins or portions thereof, thereby anchoring the transgenic to the cell membrane; or it may contain a sequence that causes the protein to accumulate in a specific region of the cell, such as a nuclear localization signal.

[0078] Non-limiting examples of transcribed sequences that may be included in the vector of the present invention include, but are not limited to: sequences expressing long transgenes; sequences expressing transgenes capable of producing different splicing variants; sequences expressing two or more transgenes (whether the two transgenes are in one transcriptional unit or in different transcriptional units); sequences expressing transgenes controlled by very long upstream and / or downstream regulatory sequences; sequences expressing short regulatory RNAs (miRNA, siRNA, etc.); sequences conferring the ability of transgenes to replicate and properly segregate in dividing cells; or combinations of the above sequences.

[0079] As described above, the vector of the present invention may include at least one transgene inserted into an HSV-1 precursor vector and operatively linked to one or more LTE and / or DNA insulator sequences. "Operationally linked" means that one or more LTE and / or DNA insulator sequences allow the transgene to be expressed in the cellular environment while the genetic elements (i.e., "genes") originally present in the HSV genome are in a transcriptionally silent state.

[0080] The transgene inserted into the vector of this invention contains at least one promoter sequence and one transcription sequence, such that the transcription sequence is regulated by the promoter. As used herein, a “promoter” refers to a DNA regulatory region capable of binding to RNA polymerase in mammalian cells and initiating transcription of an operable downstream (3' direction) sequence. For the purposes of this invention, the promoter sequence contains at least the minimum number of bases or elements required to initiate transcription of the target gene at a level higher than the background level. The promoter sequence contains a transcription start site and an RNA polymerase binding domain. Eukaryotic promoters typically (but not always) contain a “TATA” box and other DNA motifs, such as a “CAT” box or a “SP1” box.

[0081] The promoter in a transgene can be any promoter used to control / regulate the expression of a transcribed sequence. For example, the promoter can be a cell-specific or tissue-specific promoter (e.g., EOS, OCT4, Nanog (for ESC / iPSC), SOX2 (for neural stem cells), αMHC, Brachyury, Tau, GFAP, NSE, synaptophysin I (for neurons), Apo AI, albumin, ApoE (for liver), MCK, SMC α-actin, myosin heavy chain, myosin light chain (for muscle), etc.), such as promoters that specifically or preferentially control gene expression in specific cell types (e.g., hepatocytes, lung cells, epithelial cells, cardiomyocytes, nerve cells, skeletal muscle cells, embryonic stem cells, induced pluripotent stem cells or other stem cells, cancer cells, etc.).

[0082] In some embodiments, the promoter for sensory neurons includes a gene promoter encoding a sensory nerve receptor, such as transient receptor potential vanillic acid receptor 1 (TRPV1) or transient receptor potential cation channel M subfamily member 8 (TRPM8), or a gene promoter encoding a sensory nerve modulator or sensory neurotransmitter, such as a promoter of substance P, PACAP, and calcitonin gene-related peptide (CGRP). According to some embodiments, the gene promoter encoding a sensory nerve receptor according to the present invention is a promoter of the TRP gene family, more preferably a promoter of TRPV1 or TRPM8. According to some embodiments, the gene promoter encoding a sensory nerve modulator or sensory neurotransmitter according to the present invention is CGRP, or a gene promoter involved in neurite growth and stress response in sensory neurons, more preferably a gene promoter encoding adenosine diphosphate (ADVL). In other embodiments, the promoter inserted into the transgene of the present invention may be an inducible promoter.

[0083] In any of the embodiments disclosed herein, one or more transgenes may be part of one or more expression cassettes. As used herein, the term "expression cassette" refers to any nucleic acid sequence containing a promoter and a downstream coding sequence or transgene, the expression of which is driven by said promoter followed by a polyadenylation signal.

[0084] In some embodiments, the promoter inserted into the transgenic expression cassette of the vector of the present invention may be a constitutive mammalian promoter, such as promoters known in the art (e.g., EFlα, UbC, β-actin, PGK, etc.).

[0085] In addition to promoters and coding sequences, transgenes inserted into the genome of the vectors of this invention may also contain other regulatory elements. For example, transgenes may contain more than one microRNA binding site. The presence of these sites helps to downregulate transgene expression in certain cell types. Thus, for example, a vector containing a transgene intended to be specifically expressed in cancer cells or tumor cells (which may be toxic to multiple cell types) may contain a microRNA binding site for “normal” (i.e., non-malignant) cells, thereby inhibiting transgene expression in non-malignant cells.

[0086] In some embodiments, the transgene within the vector of the present invention can be monocistronic (i.e., encoding a single mRNA, producing a single protein or polypeptide) or polycistronic (i.e., encoding multiple mRNAs, producing more than one protein or polypeptide), or can express single or multiple mRNAs encoding self-cleaving polyproteins. In some embodiments, all or part of the transcribed portion of the transgene can also encode non-translated RNA, such as siRNA or miRNA. In some embodiments, the vector of the present invention can contain multiple independent monocistronic or polycistronic transgene units, each unit having its own promoter, translated or non-translated RNA sequence, and other regulatory elements.

[0087] In any of the embodiments disclosed herein, one or more target nucleic acid sequences may be introduced into various locations, such as LAT regions, one or more intergenic regions, one or more genetic regions, or combinations thereof.

[0088] LAT (Late Latency-Associated Transcript) sites or regions are repetitive sites contained in inverted repeat sequences called b and b' in the viral genome. The b and b' sequences of the viral genome are also referred to as TRL (Truncal Long Repeat) and IRL (Internal Long Repeat), respectively. In some embodiments, the HSV-1 precursor vector genome contains two LAT regions, one located in the TRL and the other in the IRL. In some embodiments, one of the LAT regions in the TRL or IRL has been deleted. In some embodiments, such as... Figure 1B As shown, when the HSV-1 precursor vector genome contains two LAT sites, exogenous DNA can be introduced into both sites in the TRL and IRL regions. In some embodiments, when the LAT site in the IRL region is deleted, exogenous DNA can only be introduced into the LAT site in the TRL region. In some embodiments, when the LAT site in the TRL region is deleted, exogenous DNA can only be introduced into the LAT site in the IRL region.

[0089] The LAT site includes an upstream DNA insulator (INS) sequence, a latent-associated promoter (LAP), a region conferring long-term expression (LTE), and a downstream DNA insulator (INS). In some embodiments, exogenous DNA is introduced between the latent-associated promoter (LAP) and the long-term expression (LTE) region (as shown in Figure 4), or between the LTE region and the DNA insulator (INS) sequence downstream of the LTE (e.g., ...). Figure 1A and 1B (As shown).

[0090] Importantly, the LAT site contains both a LTE and a DNA insulator sequence (INS), which confer the ability to express any exogenous DNA introduced into the site (e.g., therapeutic transgenes) long-term. In some embodiments, the exogenous DNA will be located between the long-term expression (LTE) motif and the downstream DNA insulator (INS) motif.

[0091] A “long-term expression sequence” or “long-term expression element (LTE)” refers to a nucleotide sequence that, when operatively linked to target exogenous DNA, enables sustained expression of a gene product for more than 15 to 45 days, 30 to 45 days, 45 to 90 days, 90 to 365 days, 365 days to several years, or even a patient’s entire life. In HSV-1, the long-term expression (LTE) sequence is identified as a region of the latent-associated transcript (LAT) originating from the LAT-associated promoter (LAP). The LTE is located downstream of the LAT transcription start site. Preferably, the LTE is located approximately 1.5 kb to approximately 3 kb downstream of the LAT transcription start site. Furthermore, DNA insulators also contribute to long-term expression. While not wishing to be confined to any particular theory, DNA insulators may suppress epigenetic silencing. Sequences conferring long-term expression, including LTE sequences and DNA insulator sequences, can be placed upstream and / or downstream of the exogenous DNA.

[0092] Those skilled in the art will recognize that other types of LTE sequences and other DNA insulator sequences have been described and are constantly being discovered. All such LTE-like sequences and DNA insulator sequences are covered within the scope of this invention.

[0093] The term “more than one target exogenous gene” as used herein may include, but is not limited to: reporter genes driven by transient promoters (e.g., GFP, RFP, luciferase, or fusion proteins, etc.), which are used for internal expression control or for biodistribution studies; recombinases driven by inducible promoters for in vivo modification of cellular or viral genes; antibiotic resistance genes, such as chloramphenicol resistance genes; elements of the tetracycline-inducible system (TRE); any exogenous DNA encoding the target gene, or combinations thereof. The use of reporter genes such as, but not limited to, cherry, RFP, GFP, or CFP, helps in identifying recombinant genomes, assessing the stability of filler fragments, and counting infectious particles (PFUs) and transduction units (TUs).

[0094] In some embodiments, the modified HSV-1 vector of the present invention contains two or more target nucleic acid sequences, which may be identical or different, and may be introduced into one or more intergenic regions (IRs) of the modified HSV-1 vector. As used herein, an intergenic region generally refers to the region between poly(A) signals of two genes in opposite directions. Several IRs have been described in viral genomes that allow the introduction of longer exogenous DNA fragments without interfering with viral replication or the expression of adjacent genes, such as IRs located between genes UL3 and UL4, genes UL7 and U8, genes UL26 and UL27, or genes UL35 and UL36. In some embodiments, one of the nucleic acid sequences is introduced into a LAT region.

[0095] In some embodiments, the modified HSV-1 vector of the present invention further comprises a third target nucleic acid sequence, which may be the same as or different from the first and / or second target nucleic acid sequences, and may be introduced into an intergenic region (IR) or a genic region. In some embodiments, the third target nucleic acid sequence is introduced into an IR different from the intergenic region (IR) of the first and second target nucleic acid sequences. In some embodiments, the third target nucleic acid sequence is introduced into a genic region.

[0096] In some embodiments, the third target nucleic acid sequence comprises exogenous or endogenous DNA, or a combination thereof, which is introduced into the modified HSV-1 vector of the present invention. In some embodiments, the third target nucleic acid sequence provides exogenous DNA introduced into the modified HSV-1 vector. In some embodiments, the third target nucleic acid sequence provides endogenous DNA introduced into the modified HSV-1 vector. In some embodiments, the third target nucleic acid sequence provides a combination of exogenous and endogenous DNA introduced into the modified HSV-1 vector.

[0097] In some implementations, introducing a third target nucleic acid sequence serves two important purposes. The first is to introduce the target DNA into the modified HSV-1 vector. The second is to adjust the size of the modified HSV-1 vector generated from the HSV-1 precursor vector to a final size of approximately 152 kb, or between 142 kb and 155 kb. In other words, the genome size of the modified HSV-1 vector generated from the HSV-1 precursor vector of this invention is comparable to that of the wild-type HSV-1 genome.

[0098] In some implementations, the third nucleic acid sequence to be introduced is selected from one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, one or more target exogenous genes (defined below), or a combination thereof.

[0099] In some embodiments, the third nucleic acid sequence is one or more HSV-1 essential genes. In some embodiments, the third nucleic acid sequence is a combination of a filler fragment as defined herein and one or more HSV-1 essential genes.

[0100] In some embodiments, the third nucleic acid sequence is one or more HSV-1 non-essential genes. In some embodiments, the third nucleic acid sequence is a combination of a filler fragment and one or more HSV-1 non-essential genes. In some embodiments, the third nucleic acid sequence is a combination of a filler fragment, one or more HSV-1 essential genes, and one or more HSV-1 non-essential genes. In some embodiments, the third nucleic acid sequence is a combination of one or more HSV-1 essential genes and one or more HSV-1 non-essential genes.

[0101] In some embodiments, the third nucleic acid sequence is one or more target exogenous genes. In some embodiments, the third nucleic acid sequence is a combination of a filler fragment and one or more target exogenous genes. In some embodiments, the third nucleic acid sequence is a combination of a filler fragment, one or more essential HSV-1 genes, and one or more target exogenous genes. In some embodiments, the third nucleic acid sequence is a combination of a filler fragment, one or more non-essential HSV-1 genes, and one or more target exogenous genes. In some embodiments, the third nucleic acid sequence is a combination of one or more essential HSV-1 genes and one or more target exogenous genes. In some embodiments, the third nucleic acid sequence is a combination of one or more non-essential HSV-1 genes and one or more target exogenous genes.

[0102] In some embodiments, when a third nucleic acid sequence is introduced into a gene region, it may be introduced into a region containing an HSV-essential gene encoding glycoprotein D (gD), thereby causing the inactivation of gD. In some embodiments, the inactivation of gD includes the deletion of the entire coding sequence of gD. The deletion of gD, along with the introduction of a third nucleic acid sequence at its location, allows the gD sequence to be replaced by another cell-targeting sequence. In some embodiments, the original gD sequence or a modified version thereof may be reintroduced via the third nucleic acid sequence.

[0103] In some implementations, the inactivation deletion of gD involves the deletion of a portion of the gD coding sequence. For example, the inactivation deletion may include the deletion of amino acids 6-38 in gD, a region primarily responsible for cell targeting. Introducing a third nucleic acid sequence into this region allows the gD cell-targeting sequence to be replaced by another cell-targeting sequence. For example, the gD cell-targeting sequence could be replaced with a scFv (single-chain antibody) targeting HER2. In some implementations, the original gD cell-targeting sequence or a modified version thereof can be reintroduced via a third nucleic acid sequence.

[0104] In some embodiments, the modified HSV-1 vector of the present invention comprises at least a fourth nucleic acid sequence; wherein the fourth nucleic acid sequence comprises one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, one or more target exogenous genes, or a combination thereof.

[0105] In some embodiments, the modified HSV-1 vector of the present invention comprises at least a fourth nucleic acid sequence; wherein the fifth nucleic acid sequence comprises one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, one or more target exogenous genes, or a combination thereof.

[0106] In some embodiments, the modified HSV-1 vector contains more than one nucleic acid sequence, which includes a DNA sequence, such as an endogenous and / or exogenous DNA sequence encoding a cell-targeting protein. This cell-targeting protein can retarget the virus to any target tissue or cell type. The cell-targeting gene used for inserting into the modified HSV-1 vector of the present invention may include, but is not limited to, HER-2, IL13α2, or modified versions thereof. For example, an example of a modified gD can be found in EP3469071 (the contents of which are incorporated herein by reference).

[0107] In any embodiment of this document, the size of one or more filler fragments will be determined based on the size of other nucleic acid sequences (e.g., one or more transgenes and / or one or more nucleic acids encoding cell-targeting proteins) introduced into the modified HSV-1 vector.

[0108] For example, if the HSV-1 precursor vector of the present invention comprises 105 kbps and introduces a transgene comprising 5 kbps and a nucleic acid comprising 3 kbps encoding a cell-targeting sequence (as described herein), then one or more filler fragments with a total length of about 25 kbps to about 55 kbps are introduced. Alternatively, in this case, one or more filler fragments with a total length of about 33 kbps to about 47 kbps are introduced. Alternatively, in this case, one or more filler fragments with a total length of about 36 kbps to about 44 kbps are introduced. Alternatively, in this case, one or more filler fragments with a total length of about 40 kbps are introduced.

[0109] For example, if the HSV-1 precursor vector of the present invention comprises 105 kbps and introduces a transgene comprising 5 kbps and a nucleic acid comprising 3 kbps encoding a cell-targeting sequence (as described herein), then one or more filler fragments of a total length of about 30 kbps to about 50 kbps are introduced. Alternatively, in this case, one or more filler fragments of a total length of about 32 kbps to about 45 kbps are introduced. Alternatively, in this case, one or more filler fragments of a total length of about 34 kbps to about 42 kbps are introduced. Alternatively, in this case, one or more filler fragments of a total length of about 39 kbps are introduced.

[0110] In any embodiment herein, the filler fragment may be introduced as a single long nucleic acid sequence into the modified HSV-1 vector of the present invention. In some embodiments, the filler fragment may be introduced as two or more filler fragments of the same or different lengths into the modified HSV-1 vector of the present invention.

[0111] In some implementations, the filler fragments can be introduced as multiple smaller filler fragments into different intergenic regions of the modified HSV-1 genome. In some implementations, these smaller filler fragments can have the same or different lengths.

[0112] It is worth noting that when the BAC sequence is present in the HSV-1 precursor vector, it can be removed during the preparation of the modified HSV-1 vector of the present invention. Therefore, after removing the BAC sequence from the final modified HSV-1 vector, the size of the BAC sequence is not considered when determining the size of more than one filler fragment, thereby making the size of the modified HSV-1 vector approximately 152 kbp.

[0113] Therefore, the preparation of any modified HSV-1 vector from the HSV-1 precursor vector will require the simultaneous construction of a cell line that can simultaneously complement the missing essential genes and optionally complement more than one of these non-essential genes.

[0114] Therefore, to facilitate the cultivation, production, and amplification of the modified HSV-1 vector of the present invention and the preparation of its stock solution, one aspect of the present invention provides a complementary cell line capable of complementing genes deleted from the HSV genome. Thus, preferred complementary cell lines according to the present invention are derived from cell types capable of naturally complementing deleted HSV genes. Such cells can be genetically engineered to express these genes using methods known in the art (e.g., by introducing expression cassettes into the cells to express genes from genetic constructs other than the HSV genome, such as cell chromosomes).

[0115] Furthermore, complementary cell lines can be genetically engineered to express genes encoding selectable markers, such as those commonly used to modify packaging cells or cells expressing any other exogenous genes. Suitable selectable genes include those conferring resistance to neomycin / G418, hygromycin, blastomycin, puromycin, bleomycin, and other similar agents.

[0116] It should be understood that those skilled in the art are familiar with methods for genetically engineering source cell types (e.g., Vero cells) to include expression constructs encoding deleted HSV proteins and other proteins (e.g., recombinases and / or selectable gene products). For example, a target gene with a selectable marker can be subcloned into a lentiviral vector, the source cells can be infected with the lentiviral vector, marker expression can be screened (e.g., blast fungicide resistance), and then the expression of the target gene can be confirmed.

[0117] The complementary cells of the present invention can be expanded and cloned. Therefore, the present invention provides a clonal population, i.e., a cell line, which comprises or is constituted, or is primarily constituted, the complementary cell line described herein.

[0118] Using the complementary cells of the present invention, the modified HSV-1 vector of the present invention can be amplified. Therefore, the present invention provides a method for amplifying the modified HSV-1 vector of the present invention. According to the method of the present invention, a complementary cell line is infected with the modified HSV-1 vector and then cultured until plaques form. The viral population is amplified by repeatedly transferring infectious particles into an ever-increasing population of fresh complementary cells. For these repeated transfers, the multiple of infection (MOI) can be between about 0.001 pfu / cell and about 0.03 pfu / cell. Finally, the vector of the present invention (in the form of packaged virus) with 90% cytopathic effect is purified from the cells.

[0119] Typically, the modified HSV-1 vector of the present invention is most effective when sufficient virus can be delivered to the cell population to ensure adequate viral exposure. Therefore, the present invention provides a viral stock, preferably a homogenized viral stock, containing the modified HSV-1 vector of the present invention. The preparation and analysis of HSV viral stocks are well known in the art. For example, the viral stock can be prepared in a rolling flask containing cells infected with the HSV-1 vector. The viral stock can then be purified using a continuous gradient and aliquoted for storage until needed. The titer of the viral stock varies considerably, depending primarily on the viral genotype, the preparation method, and the cell line used. Preferably, the viral titer of this viral stock is about 10. 6 pfu / ml, or more preferably about 10 pfu / ml. 7 pfu / ml (or at least approximately such a value). In a more preferred embodiment, the titer may be about 10. 8pfu / ml or 10 9 pfu / ml (or at least approximately such a value), while the titer is approximately 10. 10 pfu / ml or 10 11 pfu / ml or even 10 12 A high-titer stock solution with pfu / ml (or at least approximately such a value) is preferred. Therefore, according to the present invention, the titer of the HSV-1 carrier stock solution can range from about 10... 6 pfu / ml to approximately 10 12 pfu / ml variation (preferably around 10) 9 To about 10 11 (between pfu / ml).

[0120] The present invention also provides a composition comprising the HSV-1 precursor carrier of the present invention and / or a modified HSV-1 carrier, as well as a physiologically acceptable carrier. The carrier of this composition can be any suitable carrier. Preferably, the carrier is a pharmaceutically acceptable (e.g., physiologically or pharmacologically acceptable) carrier (e.g., an excipient or diluent). Pharmaceutically acceptable carriers are well known and readily available. The choice of carrier depends at least in part on the specific carrier used and the specific method of administration of the composition. The composition may also contain any other suitable components, particularly those for enhancing the stability of the composition and / or its end use. Therefore, the compositions of the present invention have a variety of suitable formulations. The following formulations and methods are merely examples and do not constitute a limitation.

[0121] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injections, which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the preparation isotonic with the target receptor's blood; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. These preparations can be packaged in single-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a lyophilized (freeze-dried) state, requiring only the addition of sterile liquid excipients (such as water for injection) before use.

[0122] Temporary formulations of injections and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets.

[0123] In addition, the composition may contain other therapeutic agents or bioactive agents. For example, it may contain therapeutic factors for treating a specific indication. Factors for controlling inflammation (such as ibuprofen or steroids) may be included as part of the composition to reduce swelling and inflammation caused by in vivo administration of the carrier and physiological stress. Immune system inhibitors may be administered using this composition to reduce any immune response against the carrier itself or related to the associated disease. Alternatively, immune enhancers may be added to the composition to upregulate the body's natural defenses against disease. Antibiotics (i.e., antimicrobial and antifungal agents) may be included to reduce the risk of infection associated with gene transfer procedures and other diseases.

[0124] Using the modified HSV-1 vector (and the stock solution and composition containing the vector) of the present invention, the present invention provides a method for expressing transgenes in nucleated cells (especially non-complementary cells). According to the present invention, the vector of the present invention is exposed to cells under suitable conditions for vector infection. Once the cells are infected, the transgene is transcribed (expressed) within the cell, provided that the promoter within the transgene is active intracellularly and the transgene is not inhibited by other regulatory mechanisms (such as microRNAs discussed herein). In other words, the vector of the present invention is used as a gene transfer and expression vector in mammalian cells.

[0125] As needed, the present invention can be used to express transgenes in cells in vivo or in vitro. For in vivo applications, the cells can be any desired cell type, such as exocrine 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 gland cells, etc.), ectodermal-derived cells (e.g., keratinized epithelial cells (e.g., cells that make up skin and hair), moist stratified barrier epithelial cells (e.g., cells of the cornea, tongue, oral cavity, gastrointestinal tract, urethra, vagina, etc.), nervous system cells (e.g., peripheral neurons and central neurons, glial cells, etc.), mesodermal-derived cells, cells of many visceral organs (e.g., kidney, liver, pancreas, heart, lung), bone marrow cells, and cancer cells within or outside tumors. Preferred and non-limiting cells suitable for infection by the vectors of the present invention include hepatocytes, lung cells, epithelial cells, cardiomyocytes, adipocytes, muscle cells, stem cells, and cancer cells.

[0126] When used in vivo, the method of the present invention can treat a disease or condition in a subject, wherein the transgene within the vector encodes one or more proteins, peptides, or other factors (e.g., non-coding RNAs (ncRNAs), such as siRNA or miRNA) with preventive or therapeutic activity. Therefore, the present invention provides a method for treating a disease or condition in a subject, comprising administering the vector of the present invention to the subject at a dose and location sufficient to infect the subject's cells, thereby causing the transgene to be expressed within the subject's cells, and wherein the transgene encodes one or more proteins, peptides, or ncRNAs with preventive or therapeutic activity. For example, the disease or condition may be a type of cancer, wherein the transgene may encode a substance that enhances tumor-killing activity (e.g., TRAIL or tumor necrosis factor (TNF)).

[0127] In other embodiments, the method of the present invention can be used in vitro to induce transgene expression in cultured cells. Similarly, the method of the present invention can be used to in vitro infect any type of cell, such as stem cells and fibroblasts, like human dermal fibroblasts (HDF) or human lung fibroblasts (HLF). Other preferred cell types for in vitro application include keratinocytes, peripheral blood mononuclear cells, hematopoietic stem cells (CD34+), or mesenchymal stem / progenitor cells. In some embodiments, the transgene encodes more than one factor that can influence cell differentiation.

[0128] In some embodiments, cells are infected in vivo or in vitro using the vector, composition, or stock solution of the present invention. These cells can be any nucleated mammalian cells for which transgene expression is desired. Therefore, the vector can be used to infect cells from a variety of mammals. It is believed that the method of the present invention can be applied to veterinary breeding therapy, for example, to express exogenous genes or compensate for defective genes in animals such as cattle, horses, sheep, goats, and pigs. Similarly, the method of the present invention can also be used for veterinary therapy of companion animals such as cats and dogs.

[0129] The modified HSV-1 vector of the present invention can also be used in the human body to express agents or factors with preventive or therapeutic activity in a medical setting. This factor (provided by the expression of one or more transgenes within the vector of the present invention) can be exogenous or a factor capable of compensating for gene defects.

[0130] In some embodiments, the present invention provides a kit comprising an HSV-1 precursor vector according to the invention and instructions for use. In some embodiments, the kit may also optionally comprise a plurality of first vials, each first vial independently containing a filler fragment of the same or different length. In some embodiments, the kit may also optionally comprise a plurality of second vials, each second vial independently containing the same or different nucleic acids encoding cell-targeting proteins.

[0131] In any embodiment of the present invention, in any HSV-1 precursor vector or modified HSV-1 vector of the present invention, the HSV-1 subspecies may be replaced by HSV-2.

[0132] The following examples further illustrate the invention, but should not be construed as limiting its scope in any way.

[0133] Example 1

[0134] When preparing the pre-HSV-1 vector according to the present invention, the gene clusters that can be deleted include, but are not limited to:

[0135] 1. Genes UL2, UL3, UL4 (10,200-12,600): 2,400nt;

[0136] 2. Genes UL10 and UL11 (23,200-25,200): 2,000 nt;

[0137] 3. Gene UL16 (30,200-31,400): 1,200nt;

[0138] 4. Genes UL20 and UL21 (40,800-43,700): 2,900 nt;

[0139] 5. Genes UL23 and UL24 (46,700-48,600): 1,900 nt;

[0140] 6. Genes UL39, UL40, UL41 (86,400-92,700): 6,300nt;

[0141] 7. Genes UL43 to UL47 (94,700-103,200): 8,500 nt;

[0142] 8. Genes UL50 and UL51 (107,700-109,100): 1,400nt;

[0143] 9. Genes UL55 and UL56 (115,400-117,100): 1,700 nt;

[0144] 10. One copy of the gene LAT, ICP0, UL34.5 (IRL) (118,700-126,100): 7,400 nt;

[0145] 11. Genes US2 to US5 (134,000-138,200): 4,200 nt; and / or

[0146] 12. Genes US7 to US12 (139.700-145.600): 5900nt.

[0147] In addition to the two copies of ICP4 (127,200–131,300 nt: 4,100 nt) and the single copy of ICP27 (113,800–115,300 nt: 1,500 nt) (thus totaling 9.7), an additional 45,800 nt of non-essential DNA may be deleted. Including these two genes, the total backbone deletion could be 9.7 + 45.8 = 55.5 kb. These figures are approximate, with an estimation error of approximately 3 kb. As an intermediate level of deletion, a less defective backbone might only lack clusters 6, 7, 10, 11, and 12 (as described above). In this case, the total deletion would be 43.5 kb.

[0148] These deletions can be introduced into the HSV-1 genome derived from the wild-type genome of HSV-1 F strain, which contains the BAC module (pBeloBAC11), which is inserted into the intergenic region between the UL3 and UL4 genes (BAC-HSV-1).

[0149] Before starting the deletion of non-essential genes, two essential genes in BAC-HSV-1 can be deleted first: ICP27 and ICP4 (two copies). The promoters of the ICP22 / ICP47 genes can be slightly modified by deleting the TAATGARAT box. Therefore, this is a non-replicating (NR) vector genome that can be cultured in Vero7b (or other complementary) cells. Furthermore, transgenes (e.g., BoNT-F or Luc or others) can be introduced into the LAT region. A single large fragment is deleted from the gene encoding ICP27, containing the entire linker region that constitutes one copy of ICP4, while the other copy of ICP4 is deleted, resulting in a 128kb HSV-1 precursor vector that does not contain the BAC module. Figure 1A ).

[0150] Insertions and / or deletions can be introduced via site-specific recombination (SSR), homologous recombination (HR), or Crisper-Cas. As described in this article, filler fragments can be introduced at the same location in deleted genes, including ICP4 and ICP27 deletions.

[0151] The missing BAC-HSV1 was then transfected into 7b cells to isolate, clone, and produce the corresponding modified HSV-1 vector.

[0152] Example 2

[0153] Two parallel strategies will be developed to introduce filler fragments into the HSV-1 vector. The first strategy aims to introduce filler fragments of different lengths into the same unique site; the second strategy aims to introduce short filler fragments into different sites.

[0154] The HSV-1 vector (hereinafter referred to as BAC-3) expressing firefly luciferase (2 kb) and still containing the BAC module (7.5 kb) located in the intergenic region between UL3 and UL4 will be used. This BAC-3 contains a long deletion (approximately 22 kb), with the first deletion extending from the start site (ICP27) of UL54 to the inner oris region (deleting UL54, 55, 56, LAT, ICP0, ICP34.5, pac, and ICP4), and the second deletion spanning the second copy of ICP4. Since this BAC-3 contains both the transgene (2 kbp) and the BAC module (7.5 kbp), the total deletion is approximately 13 kbp. After deleting the BAC module, the theoretical size of this BAC-3 in its initial state is approximately 130 kbp (20% shorter than the wild-type genome).

[0155] Example 3

[0156] This study investigated whether the genomic stability of HSV-1 vectors depends on their net genome size. The genomic stability of HSV-1 viral vectors with different genome sizes due to the deletion of non-essential viral genes was analyzed. Because it is necessary to maintain the vector size within the range of effective DNA packaging (ideally reaching 100% of the wild-type genome length), these deletions were replaced with "filler" fragments of non-coding DNA. To this end, filler fragments based on HSV-1 non-coding sequences with GC content similar to wild-type virus were designed (see Filler Fragments below). Furthermore, the filler sequences were screened to ensure they did not contain any open reading frames (ORFs).

[0157] To construct a reliable, non-toxic, and non-replicating DNA delivery platform for genomic medicine, a large portion of the HSV-1 genome must be deleted. However, these deletions result in a vector genome size below the lower limit allowed by viral packaging. Sequencing experiments using Oxford Nanopore Technology (ONT) revealed that due to backbone engineering (… Figure 3 When the genome size of the modified HSV-1 vector decreased to 131,553 bp (Figure 4a), rapid rearrangements occurred during successive passages in cell culture (Figure 5b). These rearrangements manifested as genomic instability in the viral population, resulting in high levels of genomic variation, such as amplicon generation, nucleotide substitutions, and / or genomic region duplications, after only a few passages. Therefore, these undesirable recombination events hindered the production of genomically homogeneous vector viral stock solutions. These results indicate a relatively strict limitation on the amount of packageable DNA in viral particles, and that a reduction in genome size leads to a sharp decline in viral genomic stability.

[0158] To mitigate the risk of genome rearrangement and its resulting vector instability, an HSV-1 precursor vector was constructed by inserting a non-coding sequence of a "filler" module that does not contain open reading frames or promoter regions (Fig. 4b). This modified HSV-1 vector (Fig. 4c), after transfection into mammalian cells and subsequent self-excision of the BAC module, exhibited a net genome size close to that of the wild-type virus (153,533 bp), demonstrating significantly improved genetic stability. In the modified HSV-1 vector with the filler fragment (Fig. 5a), no recombination byproducts, such as amplicon or repetitive regions of the viral genome, were observed. In contrast, the "filler-free" construct with a genome size of 131,599 bp... Figure 3 The resulting vector had an amplicon-like genome sequence of approximately 1.5 kb, containing oriS and packaging signals (Fig. 5b). These sequences were repeated multiple times, as evidenced by the overexpressed fragments (>90%) in the sequencing data, likely due to their tandem nature—the amplicon generation likely represents genomic instability due to an inefficient (shorter) vector size. Another smaller vector (138,012 bp) rearranged a repetitive region of the genome (9,000 bp) to achieve an optimal size (147,000 bp) (Fig. 5c). No such instability was observed after repeated passages in the modified HSV-1 vector with the optimal size shown in Fig. 5a.

[0159] Example 4

[0160] To further investigate the size limitations required for modified HSV-1 vectors to maintain genomic stability, several modified HSV-1 vectors of different sizes were constructed from different HSV-1 precursor vectors and filler fragments. These vectors were amplified 10-fold in Vero 7b cells, and their genomic stability was assessed. Viral genomic DNA was extracted and sequenced using Oxford nanopore technology. Analysis was performed using Geneious software. Genomes containing DNA region duplications or recombination byproduct amplicones were classified as unstable genomes. Figure 6 (Circled dots). Genomes that did not undergo recombination events were identified as stable sequences. Figure 6 (Triangle point). Recombinant byproduct amplicon and genomic duplication are associated with shorter genome size (<142kb). Figure 6 (circled dots), while stable sequences that did not undergo recombination events were associated with larger genome sizes (145kb-154kb). Figure 6 (Triangle point).

[0161] To maintain the genome size of the modified HSV-1 vector between 147 and 154 kb (regardless of transgene size), a BAC-HSV1 backbone library with decreasing filler size was constructed based on a 22 kb filler fragment. The 6 kb, 12 kb, 15 kb, or 19 kb filler sequences listed below are the decreasing-size fragments in SEQ.1, with GC content similar to wild-type HSV-1.

[0162] Non-limiting examples of sequences applicable to the present invention include the following.

[0163] SEQ.1 - Full sequence of the padded fragment (22000 bp):

[0164] GC content: 68%

[0165]

[0166] SEQ.2: 19kb filled fragment

[0167] GC content: 68%

[0168]

[0169] SEQ.3: 15kb filling fragment

[0170] GC content: 68%

[0171]

[0172] SEQ.4: 12kb filled fragment

[0173] GC content: 68%

[0174]

[0175] SEQ.5: 9kb filling fragment

[0176] GC content: 67%

[0177]

[0178] SEQ.6: 6kb filled fragment

[0179] GC content: 67%

[0180]

[0181] SEQ.7: 3kb filled fragment

[0182] GC content: 67%

[0183]

[0184] While the invention has been specifically shown and described with reference to its preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the invention as covered by the appended claims.

Claims

1. A modified HSV-1 vector comprising one or more target nucleic acid sequences and one or more filler fragments, wherein the genome size of the modified HSV-1 vector is approximately 142 kbp to approximately 160 kbp.

2. The modified HSV-1 carrier according to claim 1, wherein, The more than one target nucleic acid sequence comprises more than one target transgene, more than one nucleic acid encoding a cell-targeting protein, or a combination thereof.

3. The modified HSV-1 carrier according to claim 2, wherein, The target nucleic acid sequence contains one or more target transgenes.

4. The modified HSV-1 carrier according to claim 3, wherein, The one or more target transgenes are operatively linked to at least one sequence that confers long-term expression.

5. The modified HSV-1 carrier according to any one of claims 1 to 4, wherein, The modified HSV-1 genome is approximately 143 kbp to approximately 158 kbp in size.

6. The modified HSV-1 carrier according to claim 5, wherein, The modified HSV-1 genome is approximately 145 kbp to approximately 155 kbp in size.

7. The modified HSV-1 carrier according to claim 6, wherein, The modified HSV-1 genome is approximately 152 kbp in size.

8. The modified HSV-1 carrier according to any one of claims 1 to 7, wherein, The one or more filling fragments independently contain non-coding DNA with a GC content of 60-75%.

9. The modified HSV-1 carrier according to claim 8, wherein, The one or more filling fragments are independently selected from sequences of SEQ ID NO: 1 to SEQ ID NO:

7.

10. The modified HSV-1 carrier according to any one of claims 2 to 9, wherein, The one or more target transgenes independently constitute part of the expression cassette.

11. The modified HSV-1 carrier according to claim 10, wherein, The expression box is operatively linked to at least one sequence that imparts long-term expression.

12. The modified HSV-1 carrier according to any one of claims 4 to 11, wherein, The at least one sequence conferring long-term expression is selected from LTE and / or DNA insulators in the HSV-1 genome.

13. A method for preparing a modified HSV-1 vector, comprising: a) Remove non-essential genes, essential genes, or combinations thereof from the HSV-1 genome to obtain the HSV-1 precursor vector backbone genome of less than 130 kbp and greater than 75 kbp. b) Insert one or more target nucleic acid sequences into the HSV-1 precursor vector backbone genome; and c) Insert one or more filler fragments into the HSV-1 precursor vector backbone genome. In b) and c), the size of the modified HSV-1 vector genome is approximately 142 kbp to approximately 160 kbp.

14. The method according to claim 13, wherein, The target nucleic acid sequence comprises one or more target transgenes, one or more nucleic acids encoding cell-targeting proteins, or a combination thereof.

15. The method according to claim 14, wherein, The target nucleic acid sequence contains one or more target transgenes.

16. The modified HSV-1 carrier according to claim 15, wherein, The one or more target transgenes are operatively linked to at least one sequence that confers long-term expression.

17. The method according to any one of claims 13 to 16, wherein, Following b) and c), the size of the modified HSV-1 genome was approximately 143 kbp to approximately 158 kbp.

18. The method according to claim 17, wherein, Following b) and c), the size of the modified HSV-1 genome was approximately 147 kbp to approximately 155 kbp.

19. The method according to claim 18, wherein, Following b) and c), the size of the modified HSV-1 genome is approximately 152 kbp.

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