Improved herpes simplex virus type 1

Abstract

The present application describes an improved herpes simplex virus type 1 (HSV-1) that can be efficiently produced in a suspension cell culture, and a method of producing the HSV-1 vector in a suspension cell culture.

Description

[0001] Inventors: Grzegorz Sarek (Poland), Tomasz Benedyk (Poland) and Yohann Dickx (France) Technical Field

[0002] The present application relates to an improved herpes simplex virus type 1 (HSV-1) vector, wherein the improvement enables the improved HSV-1 vector to be efficiently produced in a suspension cell line. Background Art

[0003] Viral vectors have been used as gene transfer tools in various preclinical and clinical gene therapy applications. Among them, herpes simplex virus type 1 (HSV-1) vectors are particularly suitable for treating diseases affecting the central nervous system (CNS), such as Parkinson's disease or malignant glioma, due to their advantageous properties, including natural neurotropism, high transduction efficiency, large transgene capacity, and the ability to enter a latent state in neurons. In fact, (T-VEC, talimogene laherparepvec) consists of engineered HSV-1 and has become the first virus gene therapy product approved by the FDA for standard patient care. In addition to oncolytic HSV-1 vectors with replication ability such as T-VEC, replication-deficient HSV-1 vectors have been explored as gene therapy delivery vectors for conditions such as genetic skin diseases, pain, neuropathy, and other neurodegenerative diseases. In the past decade, research on the development of HSV-1 vectors has produced recombinant HSV-1 vectors that are non-toxic and capable of long-term transgene expression in neurons.

[0004] However, although HSV-1 vectors have become an effective and powerful treatment method, their production remains a challenging problem, limiting their speed to market. Currently, the production of HSV-1 vectors relies on adherent cell lines (such as the Vero cell line), and these cells need to be detached from their growth surface for routine maintenance, expansion culture, and counting. In adherent cell culture, cell growth is limited by the surface area, so the product yield is substantially limited. In contrast, in suspension cell culture, the infected cells used for producing viral vectors do not depend on surface growth, so expansion culture becomes much easier. Therefore, in view of the production capacity, it would be advantageous to improve HSV-1 vectors so that they can be stably produced in a suspension cell line.

[0005] As HSV-1 vectors become an increasingly attractive gene therapy vector, the demand for more efficient production processes is growing. A new generation of HSV-1 vectors that can be produced in suspension cell lines will allow for optimized production, increased vector yield, and meet the needs of various therapeutic, prophylactic, and research applications. Summary of the Invention

[0006] The present application provides a modified HSV-1 vector capable of being produced in suspension cell lines such as CHO, HeLa, H-9, Jurkat, C6 / 36, High Five, S2, Sf21, Sf9, PC-1, and HEK293. The modified HSV-1 vector comprises a mutation, wherein the mutation is a single nucleotide substitution in the US2 coding sequence or a single nucleotide substitution in the US8 coding sequence. Preferably, the single nucleotide substitution at position 827 in the US2 coding sequence results in a G276V mutation, and this position 827 is numbered relative to the position in the wild-type US2 gene (SEQ ID NO:1). More preferably, the substituted US2 coding sequence comprises the nucleic acid sequence of SEQ ID NO:2. Preferably, the single nucleotide substitution at position 348 in the US8 coding sequence results in a W116STOP mutation, and this position 348 is numbered relative to the position in the wild-type US8 gene (SEQ ID NO:3). More preferably, the substituted US8 coding sequence comprises the nucleic acid sequence of SEQ ID NO:4.

[0007] In an embodiment, the single nucleotide substitution in US8 that results in a premature stop codon can be at any other position within the region of the US8 gene that encodes the extracellular domain of the US8 protein.

[0008] In an embodiment, the single nucleotide substitution in US8 that results in a premature stop codon can be at any other position within the region of the US8 gene that encodes the transmembrane domain of the US8 protein, and this substitution results in a protein that contains a non-functional transmembrane domain.

[0009] The present application also provides a modified HSV-1 vector comprising an inactivating deletion of the US8 (gE) gene. The inactivating deletion can be a deletion within the entire coding sequence of the US8 (gE) gene or a deletion thereof, or alternatively can include a deletion of the promoter or other regulatory sequences of the gene. In one aspect, the inactivating deletion can be a complete deletion of the coding sequence of the US8 (gE) gene such that the viral genome does not contain the nucleic acid sequence of the US8 (gE) gene. As used herein, an "inactivating deletion" of the US8 gene is any deletion that results in the absence of the US8 protein on the surface of the HSV-1 virus.

[0010] In an embodiment, the inactivating deletion in the US8 gene results in a truncated protein that lacks a transmembrane domain or lacks a functional transmembrane domain.

[0011] In an embodiment, the inactivating deletion in the US8 gene results in the modified HSV-1 vector lacking the US8 protein.

[0012] Without wishing to be bound by any particular theory, it is believed that the lack of a transmembrane domain or a functional transmembrane domain in a truncated US8 protein or the absence of the US8 protein results in the lack of the US8 protein in the envelope of the modified HSV-1 virus, and the US8 protein is necessary for efficient cell-to-cell spread.

[0013] In some embodiments, the inactivating deletion comprises a single nucleotide substitution in the US8 coding sequence as described herein.

[0014] In some embodiments, prior to introducing the mutation, the parental HSV-1 vector is a wild-type HSV-1 vector, preferably the human HSV-1 F strain comprising the genome of GenBank accession number GU734771.1.

[0015] In some embodiments, prior to introducing the inactivating deletion of the US8 gene, the parental HSV-1 vector is a wild-type HSV-1 vector, preferably the human HSV-1 F strain comprising the genome of GenBank accession number GU734771.1.

[0016] In some embodiments, the term "modified HSV-1 vector" refers to an HSV-1 vector having the W116STOP mutation as described herein.

[0017] In some embodiments, the term "modified HSV-1 vector" refers to an HSV-1 vector having an inactivating deletion of the US8 gene.

[0018] In some embodiments, the modified HSV-1 vector is a recombinant HSV-1 vector, a replication-competent HSV-1 vector, a defective helper virus-independent HSV-1 vector, a helper HSV-1 vector, or an HSV-1 amplicon vector.

[0019] In some embodiments, the modified HSV-1 vector is an HSV-1 amplicon vector, wherein a helper virus-dependent packaging system is used to produce the HSV-1 amplicon vector, and wherein the helper virus-dependent system comprises a helper HSV-1 vector comprising the mutation. In some embodiments, the modified HSV-1 vector is an HSV-1 amplicon vector comprising a mutant US2 protein expressed by SEQ ID NO:2, the mutant US2 protein comprising a G276V mutation relative to the wild-type US2 protein, preferably present in the tegument of the HSV-1 amplicon vector. In some embodiments, the modified HSV-1 vector is an HSV-1 amplicon vector comprising a mutant US8 protein (gE) expressed by SEQ ID NO:4, the mutant US8 protein (gE) comprising a W116STOP mutation relative to the wild-type US8 protein (gE).

[0020] In some embodiments, the modified HSV-1 vector is an HSV-1 amplicon vector that comprises a modified US8 (gE) protein or lacks the US8 (gE) protein. In an embodiment, an inactivating deletion of the US8 gene in the helper virus can result in the presence of a non-functional US8 protein in the HSV-1 amplicon vector. In an embodiment, an inactivating deletion of the US8 gene in the helper virus can result in the lack of the US8 protein in the HSV-1 amplicon.

[0021] The modified HSV-1 vector may further comprise a genome that contains an exogenous expression cassette. The expression cassette may contain at least one nucleic acid sequence encoding a gene product.

[0022] This application also provides a pharmaceutical composition that comprises a modified HSV-1 vector as described herein and a pharmaceutical excipient.

[0023] This application also provides a kit that comprises a modified HSV-1 vector as described herein and instructions.

[0024] This application also provides a method for producing an HSV-1 vector in a suspension cell line, wherein the method comprises infecting the suspension cell line with a modified HSV-1 vector as described herein; and culturing the infected cells. This application also provides a method for producing a modified HSV-1 amplicon vector in a suspension cell line, wherein the method comprises infecting the suspension cell line with a helper virus-dependent packaging system as described herein; and culturing the infected cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Depicts wild-type HSV-1 (wt HSV-1) vector backbone and mutant HSV-1 vector backbone. The wild-type HSV-1 vector backbone contains the US2 coding sequence of SEQ ID NO:1 and the US8 coding sequence of SEQ ID NO:3. The mutant HSV-1 vector backbone contains a G276V substitution in the US2 coding sequence, resulting in the US2 coding sequence of SEQ ID NO:2; and / or a W116STOP substitution in the US8 (gE) coding sequence, resulting in the US8 coding sequence of SEQ ID NO:4.

[0026] Figure 2 Depicts a modified non-replicating HSV-1 (modified nr HSV-1) vector backbone that contains a G276V substitution in the US2 coding sequence, resulting in a US2 coding sequence of SEQ ID NO:2, and / or a W116STOP substitution in the US8 (gE) coding sequence, resulting in a US8 coding sequence of SEQ ID NO:4. "Δ" indicates deleted genes, including Δjoint and ΔICP4 (second copy).

[0027] Figure 3 Describes the parental helper HSV-1 vector backbone in a helper virus-dependent packaging system for producing HSV-1 amplicon vectors, in which two copies of the ICP4 gene have been deleted; and a modified helper HSV-1 vector backbone based on the parental helper HSV-1 vector backbone. The modified helper HSV-1 vector contains a G276V substitution in the US2 coding sequence, resulting in the US2 coding sequence of SEQ ID NO:2; and / or a W116STOP substitution in the US8(gE) coding sequence, resulting in the US8 coding sequence of SEQ ID NO:4.

[0028] Figure 4A and Figure 4B Shows that only the truncated US8 mutation W116STOP leads to an increase in the total production yield of HSV-1 vectors in HEK293 cells cultured in suspension.

[0029] Figure 5A and Figure 5B Shows that only the truncated US8 mutation W116STOP leads to an increase in the total production yield of HSV-1 vectors in HEK293 cells cultured in suspension.

[0030] Figure 6A and Figure 6B Shows that the truncated US8 mutation W116STOP does not impair the total production yield of HSV-1 vectors in the SH-SY5Y adherent cell line.

[0031] Figure 7A and Figure 7B Shows that the truncated US8 mutation W116STOP does not impair the total production yield of HSV-1 vectors in the Vero adherent cell line.

[0032] Figure 8 Shows that HSV-1tUS8 allows a greater proportion of the produced particles to be present in the supernatant. Detailed Description

[0033] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "the method" includes one or more methods and such types of steps as will be apparent to those skilled in the art upon reading this disclosure.

[0034] The term "comprising" is used interchangeably with "including", "containing", or "characterized by" and is inclusive or open-ended language that does not exclude additional, unrecited elements or method steps.

[0035] The phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the compositions and methods of the invention corresponding to the scope of each of these phrases. Thus, a composition or method that includes the recited elements or steps contemplates specific embodiments in which the composition or method consists of or consists essentially of the elements or steps.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the preferred methods and materials are now described.

[0037] As used herein, the term "subject" refers to any individual or patient on whom the method is performed. Generally, the subject is a human, but as will be understood by one of ordinary skill in the art, the subject can be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.

[0038] As used herein, "therapeutic effect" includes therapeutic and / or prophylactic benefits as described herein.

[0039] As used herein, "treating" or "treatment" refers to administering a composition to a subject or system having an undesired condition. The condition can include a disease or disorder. "Preventing" or "prevention" refers to administering a composition to a subject or system at risk of developing the condition. The condition can include susceptibility to a disease or disorder. The effect of administering the composition to the subject (whether therapeutic and / or prophylactic) can be, but is not limited to, one or more of the following: cessation of one or more symptoms of the condition, reduction or prevention of one or more symptoms of the condition, reduction in the severity of the condition, complete elimination of the condition, stabilization or delay in the development or progression of a particular event or characteristic, or minimization of the chance of occurrence of a particular event or characteristic.

[0040] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0041] The term "active fragment" refers to an amino acid fragment that is less than the entire amino acid sequence of the molecule and retains substantially the same biological activity or corresponding biological activity, for example, an activity of more than 50%, such as 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0042] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been subsequently modified, such as hydroxyproline, α-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon atom bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (such as norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics are chemical compounds that have a structure different from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid.

[0043] Amino acids can be represented herein by their common three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Nucleotides can likewise be represented by their generally accepted single-letter codes.

[0044] As used herein, a "regulatory gene" or "regulatory sequence" is a nucleic acid sequence that encodes a product (such as a transcription factor) that controls the expression of other genes.

[0045] As used herein, a "protein-coding sequence" or a sequence encoding a specific protein or polypeptide is a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into mRNA (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vitro or in vivo. The boundaries of the coding sequence are determined by the start codon at the 5' end (N-terminus) and the translation termination nonsense codon at the 3' end (C-terminus). The coding sequence can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and synthetic nucleic acids. The transcription termination sequence will typically be located at the 3' end of the coding sequence.

[0046] The term "transgene" refers to a specific nucleic acid sequence encoding an RNA and / or polypeptide or a part of a polypeptide to be expressed in a cell into which the nucleic acid sequence is introduced. The term "transgene" 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 that is naturally present in a cell into which it has been introduced; (3) a nucleic acid sequence for adding additional copies of the same (i.e., homologous) or similar nucleic acid sequence that is naturally present in a cell into which it has been introduced; or (4) a naturally occurring or homologous silenced nucleic acid sequence whose expression is induced in a cell into which it has been introduced. A "mutant form" refers to a nucleic acid sequence containing one or more nucleotides 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 contain a sequence encoding a leader peptide or a signal sequence such that the transgene product will be secreted from the cell, or the transgene may contain a leader peptide or a signal sequence and a membrane-anchoring peptide, or even a fusion protein between two naturally occurring proteins or parts thereof such that the transgene will remain anchored to the cell membrane, or a sequence that allows the protein to accumulate in a specific region of the cell, such as a nuclear localization signal.

[0047] As used herein, the term "expression cassette" or "transcription cassette" refers to an independent component of vector DNA consisting of a gene to be expressed in a transfected or transduced cell and regulatory sequences. In each successful transfection, the expression cassette directs the cellular machinery to synthesize RNA and protein. Certain expression cassettes are designed for modular cloning of protein-coding sequences such that the same cassette can be easily modified to produce different proteins. An expression cassette may consist of one or more genes and sequences controlling their expression. An expression cassette contains at least three components: a promoter sequence, an open reading frame, and a 3' untranslated region (which typically contains a polyadenylation site in eukaryotes).

[0048] As used herein, a "promoter" is defined as a regulatory DNA sequence generally located upstream of a gene that mediates the initiation of transcription by guiding the binding of RNA polymerase to DNA and initiating RNA synthesis. A promoter can be a constitutively active promoter (i.e., a promoter that is continuously in an active / "on" state), an inducible promoter (i.e., a promoter whose active / "on" or inactive / "off" state is controlled by an external stimulus, such as the presence of a specific compound or protein), a spatially restricted promoter (i.e., a transcriptional control element, enhancer, etc.; such as a tissue-specific promoter, a cell type-specific promoter, etc.), or a temporally restricted promoter (i.e., a promoter that is "on" or "off" at a specific stage of embryonic development or a specific stage of a biological process). For the purposes of the present invention, the promoter sequence comprises at least the minimum number of bases or elements required to initiate transcription of the gene of interest at a level detectably above background. The transcription start site and the RNA polymerase binding domain are contained within the promoter sequence. Eukaryotic promoters typically (but not necessarily) contain a "TATA" box and other DNA motifs, such as "CAT" or "SP1" boxes.

[0049] As used herein, the term "gene" refers to a deoxyribonucleotide sequence that contains the coding region of a structural gene. A "gene" may also include the untranslated sequences flanking the 5' and 3' ends of the coding region such that the gene corresponds to the length of the full-length mRNA. The sequence located at the 5' end of the coding region and present on the mRNA is called the 5' untranslated sequence; the sequence located at the 3' end or downstream of the coding region and present on the mRNA is called the 3' untranslated sequence. The term "gene" encompasses genes in cDNA and genomic forms. The genomic form or clone of a gene contains the coding region interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences". Introns are gene segments that are transcribed into heterogeneous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced" from the nuclear transcript or primary transcript; thus, introns are not present in messenger RNA (mRNA) transcripts. mRNA is used during translation to determine the amino acid sequence or order in a nascent polypeptide.

[0050] As used herein, the terms "functionally linked" and "operably linked" are used interchangeably and refer to the functional relationship between two or more DNA fragments, particularly the relationship between a gene sequence to be expressed and the sequence controlling its expression. For example, a promoter / enhancer sequence (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. The promoter regulatory sequence operably linked to a transcribed gene sequence is physically contiguous with the transcription sequence.

[0051] "Conservative Modification Variants" applies to amino acid sequences and nucleic acid sequences. For a particular nucleic acid sequence, conservative modification variants are nucleic acids that encode the same or substantially the same amino acid sequence, or (if the nucleic acid does not encode an amino acid sequence) nucleic acids that are substantially the same. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where a codon specifies alanine, the codon can be changed to any of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations" and are a type of conservative modification variant. Every nucleic acid sequence encoding a polypeptide herein also describes every possible silent variation of that nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to yield a molecule having the same function. Thus, every silent variation of a nucleic acid encoding a polypeptide is implicit in each such sequence.

[0052] With respect to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions, or additions of amino acids which alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservative modification variants" where the change results in the substitution of an amino acid with one having similar chemical properties. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservative modification variants are a supplement to, and not an exclusion of, the polymorphic variants, interspecies homologs, and alleles of the invention.

[0053] Each of the following eight groups consists of amino acids that are conservative substitutions for one another:

[0054] Alanine (A), Glycine (G);

[0055] Aspartic acid (D), Glutamic acid (E);

[0056] Asparagine (N), Glutamine (Q);

[0057] Arginine (R), Lysine (K);

[0058] Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0059] Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0060] Serine (S), Threonine (T); and

[0061] Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0062] Conservative substitutions (also known as conservative replacements or conservative mutations) can include substitutions such as basic amino acid for basic amino acid, acidic amino acid for acidic amino acid, polar amino acid for polar amino acid, etc. The resulting groups of amino acids may be conserved for structural reasons. These groups can be described in the form of a Venn diagram (Livingstone C.D. and Barton G.J., “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation”, Comput. Appl. Biosci., 1993, 9, 745-756; Taylor W.R., “The classification of amino acid conservation”, J. Theor. Biol., 1986, 119, 205-218), which is incorporated herein by reference).

[0063] The “percent sequence identity” is determined by comparing two optimally aligned sequences in a comparison window, wherein the polynucleotide sequence portion in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (such as the polypeptide of the present invention) that does not contain the addition or deletion, in order to achieve the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity.

[0064] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or "percent identity" refer to two or more identical sequences or subsequences. When compared in a comparison window or specified region and aligned with maximum correspondence (measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection), two sequences are "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity in the specified region, optionally 65%, 70%, 75%, 80%, 85%, 90% or 95% identity, or the above identities over the entire sequence if not specified). The present invention provides polypeptides that are substantially identical to the polypeptides exemplified herein, and their uses, including but not limited to for the treatment or prevention of neurological diseases or disorders, such as neurodegenerative diseases or disorders, and / or the treatment of spinal cord injury (SCI). Optionally, the identity exists over a region of at least about 50 nucleotides in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides in length, or over the entire length of the reference sequence.

[0065] For sequence comparison, typically one sequence is taken as the reference sequence and the test sequence is compared to it. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, optionally specifying subsequence coordinates and specifying the sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0066] As used herein, "comparison window" refers to any segment of from 20 to 600 contiguous positions, usually about 50 to about 200 contiguous positions, more usually about 100 to about 150 contiguous positions, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. For example, optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 1970, 2:482c), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 1970, 48:443), the similarity search method of Pearson and Lipman (Proc. Nat'l. Acad. Sci. USA, 1988, 85:2444), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, 1995, Suppl.).

[0067] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res., 1977, 25, 3389-3402 and Altschul et al., J. Mol. Biol., 1990, 215, 403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating a search to find longer HSPs that contain them. The word hits are extended in both directions along each sequence until the cumulative alignment score no longer increases. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops from its maximum value by an amount X; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and compares both strands. For amino acid sequences, the BLASTP program defaults to a word length of 3, an expectation value (E) of 10, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA, 89:10915), an alignment number (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and compares both strands.

[0068] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences occurred by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0069] "Nucleic acid" refers to polymers of deoxyribonucleotides or ribonucleotides and their single-stranded or double-stranded forms, and their complementary sequences. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which analogs or modifications are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to a reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNAs). In various embodiments, nucleic acids are isolated when purified from other cellular components or other contaminants (such as other nucleic acids or proteins present in the cell) by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See, e.g., F. Ausubel et al., eds., Current Protocols in Molecular Biology, 1987, Greene Publishing and Wiley Interscience, New York. In various embodiments, the nucleic acid is, for example, DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0070] As used herein, "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, and emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents.

[0071] As used herein, the term "viral vector" or "viral expression vector" refers to a nucleic acid vector that contains at least one viral genomic element and can be packaged into viral particles. In the context of the present invention, the term "viral vector" should be understood broadly to include nucleic acid vectors (such as DNA viral vectors) and the viral particles produced therefrom. In the present application, viral expression vectors include adeno-associated virus (AAV) vectors and herpes simplex virus (HSV) vectors.

[0072] The term "AAV" refers to adeno-associated virus itself or its derivatives, including recombinant AAV vector particles. In addition, as used herein, the term "AAV" includes many different serotypes that have been isolated from human and non-human primate samples. Preferred AAV serotypes are human serotypes, more preferably human AAV serotypes 2, 5, and 9, and most preferably human AAV serotype 5, which is the serotype that shows the highest level of neurotropism.

[0073] The term "herpes simplex virus (HSV)" is a complex non-integrating DNA virus that can infect a very wide range of human and animal cells. HSV includes two serotypes, herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).

[0074] As used herein, the term "replication-competent virus" refers to a virus that contains within its genome all the information necessary to permit its replication within a cell.

[0075] As used herein, the term "replication-defective viral vector" or "defective viral vector" refers to a viral vector that lacks a gene or portion of a gene necessary for successful completion of the viral life cycle to permit replication.

[0076] As used herein, the term "recombinant DNA" describes a nucleic acid molecule, i.e., a polynucleotide derived from genomic, cDNA, viral, semi-synthetic, and / or synthetic sources, which by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a virus refers to a virus that carries a recombinant genome or has been engineered to introduce a mutation, deletion, or one or more heterologous polynucleotides (including genes). The term "recombinant" as used with respect to a protein or polypeptide refers to a polypeptide produced by the expression of a recombinant nucleic acid. The term "recombinant" as used with respect to a host cell refers to a recombinant vector carrying recombinant DNA within the host cell or a cell whose genome has an insertion of recombinant DNA. The term "infection" refers to the ability of a viral vector to enter a host cell, organ, or subject, or the ability of a gene product of a viral vector to enter a host cell.

[0077] HSV-1 vector

[0078] This application provides a modified HSV-1 vector comprising a mutation, wherein said mutation permits production of the HSV-1 vector in a suspension cell line.

[0079] This application also provides a modified HSV-1 vector comprising an inactivated deletion of the US8(gE) gene.

[0080] In some embodiments, the parental HSV-1 vector is wild-type HSV-1 prior to introducing mutations into its genome. The genome size of wild-type HSV-1 is approximately 153 kbp and consists of two unique segments, UL (long unique sequence) and US (short unique sequence), each flanked by inverted repeat sequences encoding key diploid genes. It contains approximately 90 protein-coding genes and over 12 microRNAs. In a preferred embodiment, prior to introducing mutations into its genome, the parental HSV-1 vector is the human HSV-1F strain containing the genome of GenBank accession number GU734771.1. In some embodiments, the terms "modified HSV-1 vector" and "mutant HSV-1 vector" are used interchangeably in the context of "modified" and "mutant".

[0081] In some embodiments, the mutation that permits production of the modified HSV-1 vector in a suspension cell line is a single nucleotide substitution introduced into the coding sequence of US2 (SEQ ID NO:1) within the HSV-1 genome. The HSV US2 gene is non-essential for viral replication in cell culture and is predicted to encode a 291 amino acid protein of 33 kDa.

[0082] In a preferred embodiment, the mutation introduced into the coding sequence of US2 (SEQ ID NO:1) within the HSV-1 genome that permits production of the modified HSV-1 vector in a suspension cell line results in an amino acid substitution of glycine-276 to valine (G276V), numbered relative to the position in the wild-type US2 gene. In another preferred embodiment, the US2 coding sequence in the modified HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:2. In yet another embodiment, the US2 coding sequence in the modified HSV-1 vector consists of the nucleic acid sequence of SEQ ID NO:2.

[0083] In some embodiments, the mutation that permits the production of the modified HSV-1 vector in a suspension cell line is a single nucleotide substitution introduced into the coding sequence of US8 (SEQ ID NO:3) within the HSV-1 genome. The HSV-1 US8 gene (also known as the gE gene) is not essential for viral replication in cell culture and encodes a 552-amino acid protein called glycoprotein E (AKA gE). gE and gI (glycoprotein I, expressed by the HSV-1 US7 gene) tend to form a heterodimer. In epithelial cells, the gE / gI heterodimer is required for intercellular spread of the virus by sorting nascent viral particles to cell junctions. Once the virus reaches the cell junctions, the viral particles can spread very rapidly to adjacent cells by interacting with cell receptors that accumulate at these junctions (see Johnson et al., Journal of Virology, 2001, Vol. 75, No. 2, 821-833. https: / / doi.org / 10.1128 / JVI.75.2.821-833.2001). In neuronal cells, gE / gI is essential for anterograde spread of infection in the host nervous system. The gE / gI heterodimer is involved in the sorting and transport of viral structural components to the axon termini together with the US9 protein (see Snyder et al., Journal of Virology, 2008, Vol. 82, No. 21, pp. 10613-10624. https: / / doi.org / 10.1128 / JVI.01241-08).

[0084] In a preferred embodiment, the mutation introduced into the coding sequence of US8 (SEQ ID NO:3) within the HSV-1 genome that permits the production of the modified HSV-1 vector in a suspension cell line results in a premature stop codon (W116STOP) at amino acid position 116. In another embodiment, the US8 coding sequence in the modified HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:4. In yet another embodiment, the US8 coding sequence in the modified HSV-1 vector consists of the nucleic acid sequence of SEQ ID NO:4.

[0085] The suspension cell lines contemplated include, but are not limited to, CHO, HeLa, H-9, Jurkat, C6 / 36, High Five, S2, Sf21, Sf9, PC-1, and HEK293. Preferably, the suspension cell line contemplated is HEK293.

[0086] The present application also provides a modified HSV-1 vector comprising an inactivated deletion of the US8(gE) gene. The inactivated deletion can be a deletion within or of the entire coding sequence of the US8(gE) gene, or alternatively can include a deletion of the promoter or other regulatory sequences of the gene. In one aspect, the inactivated deletion can be a complete deletion of the US8(gE) gene coding sequence such that the viral genome does not contain the nucleic acid sequence of the US8(gE) gene. As used herein, an "inactivated deletion" of the US8 gene is any deletion that results in the absence of the US8 protein on the surface of the HSV-1 virus.

[0087] In some embodiments, the inactivated deletion comprises a single nucleotide substitution in the US8 coding sequence as described herein.

[0088] As used herein, "US8", "US8(gE)", and "gE" are used interchangeably and refer to the envelope glycoprotein E gene or protein, depending on the context of use.

[0089] The gE protein is a viral surface glycoprotein embedded in its envelope. Its N-terminal portion constitutes the external part of the protein, and the transmembrane domain is located between residues 420 and 440. Without wishing to be bound by any particular theory, it is believed that mutations that result in premature termination of the synthesis of the US8(gE) protein (115 amino acids versus 552 amino acids of the native protein) may result in the complete absence of the protein in the mutant virus stock.

[0090] Attenuated HSV-1 vector

[0091] In an embodiment, the modified HSV-1 vector as described herein is an attenuated HSV-1 vector.

[0092] The present application also provides an attenuated HSV-1 vector comprising a mutation as described herein. The mutation allows for the production of the attenuated HSV-1 vector in a suspension cell line.

[0093] The present application also provides an attenuated HSV-1 vector comprising an inactivated deletion of the US8 gene. The inactivated deletion allows for the production of the attenuated HSV-1 vector in a suspension cell line.

[0094] As used herein, the terms "attenuated" and "replication-competent" are used interchangeably and both describe vectors derived from attenuated viruses in which non-essential genes have been mutated or deleted. Removal of one or more non-essential genes can reduce pathogenicity without the need for a cell line to supplement growth. Replication-competent HSV-1 vectors with mutations in genes affecting viral replication, neurovirulence, and immune evasion have been developed and tested for safety and efficacy in multiple mouse models.

[0095] As used herein, the qualifier "essential" in the expression "essential gene" or "non-essential gene" refers to whether a given gene is necessary for the replication and packaging of the viral genome to produce infectious progeny virus particles. HSV-1 essential genes include UL1, 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), ICP0 (2 copies), LAT (2 copies), UL2-UL4, UL10, UL11, UL13, UL16, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43-UL47, UL50, UL51, UL55, UL56, US1-US5, US7-US12.

[0096] In any embodiment of obtaining an attenuated HSV-1 vector as described herein by mutating or deleting non-essential genes, at least one of the two non-essential genes, US2 and US8, is retained in the attenuated HSV-1 genome to introduce a mutation as described herein.

[0097] In some embodiments, the mutation that allows production of the attenuated HSV-1 vector in a suspension cell line is a single nucleotide substitution introduced into the coding sequence of US2 (SEQ ID NO:1) within the attenuated HSV-1 genome.

[0098] In a preferred embodiment, the mutation introduced into the coding sequence of US2 (SEQ ID NO:1) within the attenuated HSV-1 genome that allows production of the attenuated HSV-1 vector in a suspension cell line results in an amino acid substitution of glycine-276 to valine (G276V), numbered relative to the position in the wild-type US2 gene. In another preferred embodiment, the US2 coding sequence in the attenuated HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:2. In yet another embodiment, the US2 coding sequence in the attenuated HSV-1 vector consists of the nucleic acid sequence of SEQ ID NO:2.

[0099] In some embodiments, the mutation that allows production of the attenuated HSV-1 vector in a suspension cell line is a single nucleotide substitution introduced into the coding sequence of US8 (SEQ ID NO:3) within the HSV-1 genome.

[0100] In a preferred embodiment, the introduction of the US8 (SEQ ID NO:3) coding sequence into the attenuated HSV-1 genome, a mutation that allows the production of the attenuated HSV-1 vector in a suspension cell line results in a premature stop codon (W116STOP) at amino acid position 116. In another embodiment, the US8 coding sequence in the attenuated HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:4. In yet another embodiment, the US8 coding sequence in the attenuated HSV-1 vector consists of the nucleic acid sequence of SEQ ID NO:4.

[0101] In some embodiments, the inactivating deletion of the US8 gene allows the production of the attenuated HSV-1 vector in a suspension cell line.

[0102] The attenuated HSV-1 vectors contemplated herein include any clinically available, under development and testing, or in research and development attenuated HSV-1 vectors. The attenuated HSV-1 vectors also include any attenuated HSV-1 vectors that do not exist at the time of the present invention but will be developed in the future.

[0103] In some embodiments, the attenuated HSV-1 vectors contemplated herein may be oncolytic HSV-1 vectors that preferentially infect and kill tumor cells. Non-limiting examples of oncolytic HSV-1 vectors useful in this application include hrR3 (ICP6-deficient), R3616 (γ34.5-deleted), G207 (γ34.5-deleted and ICP6-deficient), HSV1716 (γ34.5-deleted), T-VEC (talimogene laherparepvec, γ34.5-deleted and ICP-deleted). The oncolytic HSV-1 vectors useful in this application may be selected from those described by Peters et al. in Molecular Therapy–Oncolytics, 2015, 2, 15010 (https: / / doi.org / 10.1038 / mto.2015.10).

[0104] The suspension cell lines contemplated include, but are not limited to, CHO, HeLa, H-9, Jurkat, C6 / 36, High Five, S2, Sf21, Sf9, PC-1, and HEK293. Preferably, the suspension cell line contemplated is HEK293.

[0105] Defective HSV-1 vector

[0106] In an embodiment, the modified HSV-1 vector as described herein is a defective HSV-1 vector.

[0107] The present application also provides a defective HSV-1 vector comprising a mutation as described herein. The mutation allows for the production of the defective HSV-1 vector in a suspension cell line.

[0108] The present application also provides a defective HSV-1 vector comprising an inactivated deletion of the US8 gene as described herein. The inactivated deletion allows for the production of the defective HSV-1 vector in a suspension cell line.

[0109] As used herein, the terms "defective" and "non-replicating" in the expressions "defective HSV-1" and "non-replicating HSV-1" are used interchangeably and both refer to a vector derived from a defective virus in which essential genes required for viral replication have been mutated or deleted. These deletions significantly reduce the cytotoxicity of the viral vector by preventing early and late viral gene expression and, together with other deletions involving "non-essential" genes, also create space for the introduction of unique expression cassettes for independent regulation of different transgenes. In gene therapy applications where co-expression of multiple gene products is required, the use of these defective vectors can easily achieve therapeutic effects.

[0110] The defective HSV-1 vector contemplated can be a helper virus-independent defective HSV-1 vector.

[0111] In some embodiments, the genome of the helper virus-independent defective HSV-1 vector in the present application can comprise at least a complete deletion of the genes encoding two essential proteins, ICP4 and ICP27. The ICP4 gene has two copies, located in the inverted repeat sequences (referred to as c and c', i.e., short internal repeat sequences and short terminal repeat sequences) of the viral genome, and both copies of the gene are deleted. The gene encoding ICP27 (UL54) is located in the unique long (UL) sequence of the viral genome. The defective HSV-1 vector can also lack other genes encoding non-essential proteins, such as ICP34.5, UL55, UL56, and / or UL41 proteins, and carry an expression cassette (preferably in the LAT (latency-associated transcript) region of the vector genome) embedded in the vector genome.

[0112] In some embodiments, the defective HSV-1 vector further lacks one copy of the ICP0 gene. In a preferred embodiment, one copy of the IPC0 gene is removed from the LAT, ICP0, UL34.5 cluster in the IRL (long internal repeat sequence) region of the HSV vector.

[0113] In some embodiments, the vector according to the present invention is a defective vector carrying the expression cassette as described herein, and the expression cassette is driven by a promoter described in other parts of this document.

[0114] In any embodiment of obtaining a defective HSV-1 vector as described herein by mutating and / or deleting essential and / or non-essential genes, at least one of the two non-essential genes US2 and US8 is retained in the defective HSV-1 genome to introduce a mutation as described herein.

[0115] In some embodiments, the mutation that allows the production of the defective HSV-1 vector in a suspension cell line is a single nucleotide substitution introduced into the coding sequence of US2 (SEQ ID NO:1) within the defective HSV-1 genome.

[0116] In a preferred embodiment, the mutation introduced into the coding sequence of US2 (SEQ ID NO:1) within the defective HSV-1 genome that allows the production of the defective HSV-1 vector in a suspension cell line results in an amino acid substitution of glycine-276 to valine (G276V), numbered relative to the position in the wild-type US2 gene. In another preferred embodiment, the US2 coding sequence in the defective HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:2. In yet another embodiment, the US2 coding sequence in the defective HSV-1 vector consists of the nucleic acid sequence of SEQ ID NO:2.

[0117] In some embodiments, the mutation that allows the production of the defective HSV-1 vector in a suspension cell line is a single nucleotide substitution introduced into the coding sequence of US8 (SEQ ID NO:3) within the HSV-1 genome.

[0118] In a preferred embodiment, the mutation introduced into the coding sequence of US8 (SEQ ID NO:3) within the defective HSV-1 genome that allows the production of the defective HSV-1 vector in a suspension cell line results in a premature stop codon (W116STOP) at amino acid position 116. In another embodiment, the US8 coding sequence in the defective HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:4. In yet another embodiment, the US8 coding sequence in the defective HSV-1 vector consists of the nucleic acid sequence of SEQ ID NO:4.

[0119] In some embodiments, an inactivating deletion of the US8 gene allows the production of the defective HSV-1 vector in a suspension cell line.

[0120] The defective HSV-1 vectors contemplated herein include any clinically available, developing, testing, or in research and development defective HSV-1 vectors. The defective HSV-1 vectors also include any defective HSV-1 vectors that do not exist at the time of the present invention but will be developed in the future.

[0121] Non-limiting examples of the defective HSV-1 vectors useful in the present application include HSV-1d106S, KOS strain 5dl1.2, 17D30EBA strain, KOS strain d120, and NP2. The defective HSV-1 vector used in the present application can be selected from the vectors described in US Patent US11414666B2, the vectors described in US Application No. 63 / 284,176, or the vectors described in French Application FR2212771. In some embodiments, the defective HSV-1 vector used in the present application can be a "pre-HSV-1 vector" described in US Application No. 63 / 284,176 or French Application FR2212771, in which non-essential genes, essential genes, or a combination thereof have been deleted to obtain a genome comprising less than 130 kbp and greater than 75 kbp.

[0122] Suspension cell lines contemplated include, but are not limited to, CHO, HeLa, H-9, Jurkat, C6 / 36, High Five, S2, Sf21, Sf9, PC-1, and HEK293. Preferably, the suspension cell line contemplated is HEK293.

[0123] The defective HSV-1 vector contemplated can be a defective HSV-1 vector that is dependent on a helper virus, i.e., an HSV-1 amplicon vector.

[0124] "Amplicon" or "amplicon vector" refers to a defective vector that is dependent on a helper virus and whose genome lacks most or all of the HSV genes encoding viral proteins. The genome of an amplicon vector is a concatemer DNA composed of multiple tandem copies of a plasmid, and the plasmid (referred to as an amplicon plasmid) carries a DNA replication origin and a packaging signal from the HSV-1 genome in addition to the transgene DNA of interest (i.e., an expression cassette). As used herein, "dependent on a helper virus" refers to a viral vector that depends on the help of a helper virus for replication.

[0125] In any embodiment where the defective HSV-1 vector is an HSV-1 amplicon vector, a helper virus-dependent packaging system is used to produce the HSV-1 amplicon vector, wherein the helper virus-dependent packaging system comprises a helper HSV-1 vector and the mutations as described herein are introduced into the genome of the helper HSV-1 vector. In some embodiments, the helper HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:2 in the US2 coding sequence region. In some embodiments, the helper HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:4 in the US8 (gE) coding sequence region. In some embodiments, the helper HSV-1 vector comprises the nucleic acid sequence of SEQ ID NO:2 in the US2 coding sequence region and the nucleic acid sequence of SEQ ID NO:4 in the US8 (gE) coding sequence region.

[0126] In some embodiments, the helper HSV-1 vector contains an inactivating deletion within the US8 (gE) gene.

[0127] In some embodiments, the HSV-1 amplicon vector may not contain a nucleic acid sequence comprising a mutation as described herein, but may contain a protein comprising a mutation as described herein, preferably on the surface of the HSV-1 amplicon vector envelope or in the tegument of the HSV-1 amplicon. In some embodiments, the HSV-1 amplicon vector may contain a mutant US2 protein expressed by SEQ ID NO:2, which contains a G276V mutation relative to the wild-type US2 protein. In some embodiments, the HSV-1 amplicon vector may contain a mutant US8 protein (gE) expressed by SEQ ID NO:4, which contains a W116STOP mutation relative to the wild-type US8 protein (gE). In some embodiments, the HSV-1 amplicon vector may contain a mutant US2 protein (containing a G276V mutation relative to the wild-type US2 protein) expressed by SEQ ID NO:2 and a mutant US8 protein (gE, containing a W116STOP mutation relative to the wild-type US8 protein (gE)) expressed by SEQ ID NO:4.

[0128] In some embodiments, the HSV-1 amplicon vector may contain an inactivating deletion of the US8 (gE) protein. In some embodiments, the modified HSV-1 vector is an HSV-1 amplicon vector that contains a modified US8 (gE) protein or lacks the US8 (gE) protein. In an embodiment, an inactivating deletion of the US8 gene in the helper virus can result in the presence of a non-functional US8 protein in the HSV-1 amplicon vector. In an embodiment, an inactivating deletion of the US8 gene in the helper virus can result in the absence of the US8 protein in the HSV-1 amplicon vector.

[0129] In any embodiment where the defective HSV-1 vector is an HSV-1 amplicon vector, the mutations as described herein are introduced into the genome of the helper HSV-1 vector in the helper virus-dependent packaging system used to produce the HSV-1 amplicon vector. As a non-limiting example, Zaupa et al. described an improved packaging system that uses Cre-loxP site-specific recombination to delete the packaging signal of the defective helper HSV-1 vector genome, thereby producing high levels of non-cytotoxic HSV-1 amplicon vectors (see Human Gene Therapy. July 2003, 1049-1063. http: / / doi.org / 10.1089 / 104303403322124774). As Figure 3As shown, the helper HSV-1 vector of Zaupa et al. is used herein as an exemplary parental helper HSV-1 vector for introducing the mutations of the present application.

[0130] Additional genetic modifications

[0131] In some embodiments, the modified HSV-1 vectors as described herein (i.e., HSV-1 vectors, attenuated HSV-1 vectors, and defective HSV-1 vectors, each having the mutations as described herein) may further comprise additional genetic modifications, such as the insertion of one or more transgenes of interest or the deletion of one or more nucleotide sequences.

[0132] In some embodiments, the modified HSV-1 vector may further comprise a genome containing an exogenous expression cassette. The expression cassette may comprise at least one nucleic acid sequence encoding a gene product (i.e., at least one transgene of interest). Preferably, the genome containing the exogenous expression cassette may be introduced into the LAT locus, which is a repetitive locus contained within the inverted repeat sequences (referred to as b and b') of the viral genome. The b and b' sequences of the viral genome are also referred to as TRL (long terminal repeat) and IRL (long internal repeat), respectively. In some embodiments, the viral genome contains two LAT regions, one 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, when the vector genome contains two LAT loci, the genome containing the exogenous expression cassette may be introduced into both loci in the TRL region and the IRL region. In some embodiments, when the LAT locus in the IRL region is deleted, the genome containing the exogenous expression cassette may be introduced only into the LAT locus in the TRL region. In some embodiments, when the LAT locus in the TRL region is deleted, the genome containing the exogenous expression cassette may be introduced only into the LAT locus in the IRL region.

[0133] The LAT locus includes an upstream DNA insulator (INS) sequence, a latency-associated promoter (LAP), a region conferring long-term expression (LTE), and a downstream DNA insulator (INS). In some embodiments, the genome containing the exogenous expression cassette is introduced between the latency-associated promoter (LAP) and the long-term expression (LTE) region, or between the LTE region and the DNA insulator (INS) sequence downstream of LTE.

[0134] In a preferred embodiment, the genome containing the exogenous expression cassette is placed between the latency-associated promoter (LAP) and the long-term expression (LTE) region (site 1), or between the LTE region and the DNA insulator (INS) sequence downstream of LTE (site 2). In some embodiments, one or more HSV-1 non-essential genes are introduced into the modified HSV-1 vector.

[0135] As described herein, various transgenic genes of interest can be introduced into the exogenous expression cassette of the modified HSV-1 vector. The modified HSV-1 vector can be used to express any viral, bacterial, or cancer gene product. For example, the transgenic gene of interest can encode a gene product capable of inhibiting / silencing neurotransmission (e.g., wild-type or modified botulinum neurotoxin light chain, antisense RNA (AS-RNA) targeting SNARE proteins, GAD67, GAD65, ribosome-inactivating proteins (RIPs), and / or neurotoxin receptors (NTRs)). For example, the transgenic gene of interest can encode a gene product capable of inducing a protective immune response (e.g., the SARS-CoV-2 (COVID-19) spike protein).

[0136] In some embodiments, any modified HSV-1 vector as described herein can simultaneously contain a G276V mutation relative to the wild-type US2 coding sequence and a W116STOP mutation relative to the wild-type US8 coding sequence. In some embodiments, the HSV-1 vector, attenuated HSV-1 vector, and defective HSV-1 vector (each having the mutations as described herein) can simultaneously contain the US2 gene with the nucleic acid sequence of SEQ ID NO:2 and the US8 gene with the nucleic acid sequence of SEQ ID NO:4.

[0137] In some embodiments, any modified HSV-1 vector as described herein can contain a W116STOP mutation relative to the wild-type US8 coding sequence. In some embodiments, the HSV-1 vector, attenuated HSV-1 vector, and defective HSV-1 vector (each having the mutations as described herein) can contain the US8 gene with the nucleic acid sequence of SEQ ID NO:4.

[0138] In some embodiments, any modified HSV-1 vector as described herein can contain an inactivating deletion of the US8 gene. In some embodiments, the HSV-1 vector, attenuated HSV-1 vector, and defective HSV-1 vector (each having the mutations as described herein) can contain an inactivating deletion in the US8 gene.

[0139] Pharmaceutical composition

[0140] The improved HSV-1 vector described in the present application can be administered in a pharmaceutical composition, which may comprise a pharmaceutically acceptable carrier. The carrier of the composition can be any suitable carrier for the vector. The carrier is typically liquid, but can also be solid, or a combination of liquid and solid components. The carrier is desirably a pharmaceutically acceptable (e.g., physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). The composition may further comprise any other suitable components, especially components for enhancing the stability of the composition and / or its end use. Accordingly, there are various suitable formulations for the viral vector composition. The following formulations and methods are merely exemplary and in no way limiting.

[0141] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may comprise suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers (such as ampoules and vials), and may be stored under lyophilized (freeze-dried) conditions, requiring only the immediate addition of a sterile liquid excipient (such as water for injection) prior to use. Temporary injection solutions and suspensions can be prepared from the previously described sterile powders, granules, and tablets.

[0142] In addition, the composition may comprise additional therapeutic or bioactive agents. For example, there may be therapeutic factors for treating specific indications. Factors that control inflammation (such as ibuprofen or steroids) can be part of the composition to reduce swelling, inflammation, and physiological discomfort associated with in vivo administration of the viral vector. Immunosuppressive agents can be co-administered with the composition to reduce any immune response to the vector itself or to the condition. Alternatively, immunostimulants can be included in the composition to upregulate the body's natural defenses against the disease. Antibiotics (i.e., microbicides and fungicides) can be present to reduce the risk of infection associated with gene transfer procedures and other conditions.

[0143] Method for manufacturing HSV-1 vector in suspension cell line

[0144] The present application also provides a method for producing an improved HSV-1 vector in a suspension cell line, wherein the method comprises infecting the suspension cell line with the improved HSV-1 vector as described herein; and culturing the infected cells.

[0145] As described herein, the modified HSV-1 vector comprises a mutation selected from: a single nucleotide substitution introduced into the US2 coding sequence and a single nucleotide substitution introduced into the US8 coding sequence. In one embodiment, the single nucleotide substitution introduced into the US2 coding sequence results in a G276V amino acid substitution, which is numbered relative to the position in the wild-type US2 gene (SEQ ID NO:1). In another embodiment, the single nucleotide substitution introduced into the US8 coding sequence results in a premature stop codon (W116STOP), which is numbered relative to the position in the wild-type US8 gene (SEQ ID NO:3).

[0146] As described herein, the modified HSV-1 vector comprises a mutation selected from a single nucleotide substitution introduced into the US8 coding sequence. In one embodiment, the single nucleotide substitution introduced into nucleotide position 348 of the US8 coding sequence results in a premature stop codon (W116STOP), which is numbered relative to the position in the wild-type US8 gene (SEQ ID NO:3).

[0147] As described herein, the modified HSV-1 vector comprises an inactivating deletion of the US8 coding sequence.

[0148] The parental HSV-1 vector relative to the modified HSV-1 vector can be selected from a wild-type HSV-1 vector (preferably the human HSV-1F strain comprising the genome of GenBank accession number GU734771.1), a recombinant HSV-1 vector, an attenuated HSV-1 vector, a non-replicating HSV-1 vector, a defective and helper virus-independent HSV-1 vector.

[0149] The present application also provides a method for producing a modified HSV-1 amplicon vector in a suspension cell line, wherein the method comprises infecting the suspension cell line with a helper virus-dependent packaging system comprising an HSV-1 amplicon vector or an HSV-1 amplicon plasmid and a modified helper HSV-1 vector comprising a mutation as described herein; and culturing the infected cells. In some embodiments, the resulting HSV-1 amplicon vector produced by the above method may comprise a mutant US2 protein expressed by SEQ ID NO:2, which comprises a G276V mutation relative to the wild-type US2 protein. In some embodiments, the resulting HSV-1 amplicon vector produced by the above method may comprise a mutant US8 protein (gE) expressed by SEQ ID NO:4, which comprises a W116STOP mutation relative to the wild-type US8 protein (gE). In some embodiments, the resulting HSV-1 amplicon vector produced by the above method may comprise a mutant US2 protein expressed by SEQ ID NO:2 (comprising a G276V mutation relative to the wild-type US2 protein) and a mutant US8 protein (gE, comprising a W116STOP mutation relative to the wild-type US8 protein (gE)) expressed by SEQ ID NO:4. In a preferred embodiment, the mutant US2 protein is present in the tegument of the HSV-1 amplicon.

[0150] In some embodiments, due to an inactivating deletion within the US8 (gE) gene in the helper virus, the resulting HSV-1 amplicon vector produced by the above method may lack the US8 protein.

[0151] The parental HSV-1 vector contemplated may be or be derived from any HSV-1 strain described herein. Parental HSV-1 vectors include any clinically available, developing, and tested or in research and development HSV-1 vectors. Parental HSV-1 vectors include any HSV-1 vectors that do not exist at the time of the present invention but will be developed in the future. As used herein, a modified HSV-1 vector is a parental HSV-1 vector comprising one or two mutations as described herein.

[0152] The present application also provides a method for producing an HSV-1 vector in a suspension cell line, wherein the HSV-1 vector is modified and comprises a US2 gene having the nucleic acid sequence of SEQ ID NO:2 and / or a US8 gene having the nucleic acid sequence of SEQ ID NO:4. The HSV-1 vector is selected from wild-type HSV-1 vectors (such as the human HSV-1F strain), replication-competent HSV-1 vectors, defective non-helper virus-dependent HSV-1 vectors, and HSV-1 amplicon vectors.

[0153] The present application also provides a method for producing HSV-1 vectors in a suspension cell line, wherein the HSV-1 vectors are modified and contain an inactivated deletion of the US8 gene. The HSV-1 vectors are selected from wild-type HSV-1 vectors (such as the human HSV-1 F strain), replication-competent HSV-1 vectors, defective helper virus-independent HSV-1 vectors, and HSV-1 amplicon vectors.

[0154] The suspension cell lines considered include, but are not limited to, CHO, HeLa, H-9, Jurkat, C6 / 36, High Five, S2, Sf21, Sf9, PC-1, and HEK293. Preferably, the suspension cell line considered is HEK293.

[0155] In some embodiments, when the HSV-1 vector is a defective HSV-1 vector, the suspension cell line used in the method of the present invention can simultaneously express ICP4 and ICP27 proteins.

[0156] The following examples are intended to illustrate and not limit the present invention. All documents cited in this disclosure are hereby expressly incorporated by reference.

[0157] Examples

[0158] Example 1

[0159] Screening for HSV-1 mutants suitable for production in suspension cells

[0160] The original HSV-1 wild-type virus stock (P0) was generated by transiently transfecting a bacterial artificial chromosome (BAC) carrying the full sequence of the HSV-1 F strain into Vero 7b cells using Lipofectamine 2000 transfection reagent. By co-transfection with a plasmid encoding Cre recombinase, the BAC element flanked by loxP recombination sites was ensured to be excised from the genome. The resulting virus stock was harvested, and the titer was determined using the plaque assay on Vero 7b cells.

[0161] HSV-1 wild-type virus stocks H1 to H10 were generated by subsequent rounds of infection of suspension HEK293F cells (reference A35347). To obtain HSV-1 wild-type H1, 30 × 10 6 cells were centrifuged at 300 g for 5 minutes, resuspended in 5 mL LV-MAX TMIn the production medium, HSV-1 wild-type P0 was infected at an MOI of 0.05. After incubation on a roller shaker for 90 minutes, the infected cells were transferred to a 125 mL conical flask (reference 431143), supplemented with medium to 30 mL, and cultured with shaking at 130 rpm at 37 °C for 96 hours (reference 88881102). To harvest the virus, dextran sulfate at a final concentration of 0.33 M, sodium chloride at 73 μg / mL, and DNase (reference D4527-20KU) were added to the medium, followed by incubation at 37 °C for 4 hours. Cell debris was precipitated by centrifugation at 2,000 g for 5 minutes at 4 °C and discarded, and the supernatant was filtered through a 0.45 μm PES filter (reference 257201). The virus was then precipitated by centrifugation at 21,000 g for 1 hour at 4 °C and resuspended in 1 mL of PBS. The resulting HSV-1 H1 stock solution was then titrated by plaque assay and used for subsequent infection rounds to obtain stock solutions H2, etc.

[0162] Stock solution P1 was obtained by the above method, except that Vero 7b cells cultured in DMEM (reference 41965-039) supplemented with 10% FBS (reference 10500-064) were used.

[0163] Genomic DNA was then isolated from the purified virus stock solution using a genomic DNA extraction kit. The isolated DNA samples were used to prepare libraries using the Oxford Nanopore Rapid Barcoding Kit (SQK-RBK110-96) and sequenced using a FLO-MIN106 flow cell and a MinION Mk1B sequencing device. Base calling was performed in high-accuracy mode using MinKNOW GUI 5.3.6, and reads shorter than 200 bp or with a total score lower than 9 were filtered out. Using Geneious 2022.2.2 software, the resulting sequencing data was analyzed using the "Map to Reference Sequence" function (using the Minimap 2.24 mapping algorithm). Using the built-in "Find Variations / SNPs" function, variations were identified with the following parameters: minimum variation frequency 0.05, maximum variation p-value 10 -6 , find variations only within the CDS, and the homopolymer quality was reduced by 50%.

[0164] Insertion-deletion mutations that cause frameshifts and variations in homopolymer length were excluded. From the obtained variations, those that increased in frequency in subsequent passages H1 to H10 and were completely absent in passage P1 were selected for further verification. The two identified mutations involved:

[0165] 1) A single nucleotide substitution in the US2 coding sequence, resulting in a change from glycine-276 to valine, and

[0166] 2) A single nucleotide substitution in the coding sequence of US8 (also known as glycoprotein E) results in the formation of a premature stop codon at position 116, thereby truncating the protein (the original length of US8 is 552 amino acids).

[0167] Table 1. Identified mutations

[0168]

[0169]

[0170] Example 2: gE (US8) mutation increases the production yield of HSV in HEK293 suspension cell line

[0171] To evaluate the effects of these two mutations, US2 and US8, on the production yield in HEK293 suspension cell line, three recombinant HSV-1 vectors were designed from wild-type HSV-1:

[0172] HSV-1mUS2: Carrying only the US2_G276V mutation

[0173] HSV-1tUS8: Carrying only the US8_W116STOP mutation

[0174] HSV-1mUS2_tUS8: Carrying both the US2_G276V and US8_W116STOP mutations

[0175] These recombinant HSV-1 vectors were respectively produced in HEK293F cells (reference A35347) in 6-well plates at a multiplicity of infection of 0.03 and a cell density of 1×10 6 cells / mL, 2 mL per well, with technical replicates. The produced vectors were harvested, and the titers were determined using the plaque assay on Vero7B cells at 24 hours, 48 hours, 72 hours, and 96 hours.

[0176] As Figure 4A and 4B shown, only the truncated US8 mutation W116STOP results in an increase in the production yield of HSV-1 vectors in HEK293 suspension cells.

[0177] Example 3: gE (US8) mutation increases the production yield of HSV in HEK293 suspension cell line

[0178] To further evaluate the effect of the US8 mutation on the production yield in HEK293 suspension cell line, wild-type HSV-1 and the HSV-1tUS8 vector were used to infect HEK293F cells in Erlenmeyer flasks.

[0179] These recombinant HSV-1 vectors were produced separately in HEK293F cells (reference A35347) in 125 mL vented-cap conical flasks at a multiplicity of infection of 0.05, with a final volume of 30 mL and a cell density of 1×10 6 cells / mL, with biological triplicates. The produced vectors were harvested and titers were determined using plaque assays on Vero 7B cells at 24 hours, 48 hours, 72 hours, and 96 hours.

[0180] As Figure 5A and 5B shown, only the truncated US8 mutant W116STOP resulted in increased production yields of HSV-1 vectors in HEK293 suspension cells.

[0181] Example 4: The effect of gE(US8) mutations on production efficiency is related to the spread of HSV particles

[0182] To evaluate the conditions for the improved production efficiency obtained from gE mutations, two experiments were conducted in which wild-type HSV-1 and HSV-1tUS8 were cultured in two different cell lines:

[0183] the adherent Vero cell line; and

[0184] the adherent SH-SY5Y cell line.

[0185] These recombinant HSV-1 vectors were produced separately in each cell line, the produced vectors were harvested, and titers were determined using plaque assays on Vero 7B cells at 24 hours, 48 hours, 72 hours, and 96 hours.

[0186] For suspension cells, the vectors were produced in 125 mL vented-cap conical flasks at a multiplicity of infection of 0.05, with a total volume of 30 mL and a cell density of 1×10 6 cells / mL, with biological triplicates.

[0187] For adherent cell lines, the virus was produced in 24-well plates at a multiplicity of infection of 0.03, with 1×10 5 cells per well, with technical replicates.

[0188] According to the results, the increased yield of HSV-1tUS8 is related to the suspension state of the cells in the medium rather than the cell type (Figures 6 and 7). These results also demonstrate that the US8 W116 STOP mutation does not impair the replication or infectivity of the modified HSV-1 vector in other cell types.

[0189] Example 5: gE(US8) mutations increase the proportion of HSV-1 vectors available in the supernatant

[0190] Titration of the produced HSV-1 particles was performed based on the recovery of particles in the supernatant and in the cells. From a production perspective, the recovery of particles from the cells requires cell lysis (which can be achieved by different methods known in the art), which further increases the purification requirements to obtain a high-quality HSV-1 product formulation.

[0191] In the HEK293 cell line (clone 5B8), the proportion of HSV-1 tUS8 particles in the supernatant was studied and compared with the total particles produced.

[0192] The virus production method was as described in Example 2, except that for each collection time point, in addition to 200 μL of the batch culture, another 200 μL of the culture sample was taken and centrifuged at 2000 g for 5 minutes. The titers of the different fractions were determined by plaque assay using Vero7B cells.

[0193] As Figure 8 shown, HSV-1 tUS8 caused a larger proportion of the produced particles to be present in the supernatant.

[0194] SEQ ID NO:1

[0195] DNA

[0196] HSV-1 US2

[0197]

[0198] SEQ ID NO:2

[0199] DNA

[0200] Artificial sequence HSV-1US2_G276V

[0201]

[0202]

[0203] SEQ ID NO:3

[0204] DNA

[0205] HSV-1 US8

[0206]

[0207]

[0208] SEQ ID NO:4

[0209] DNA

[0210] Artificial sequence HSV-1US8_W116STOP

[0211]

Claims

1. A modified HSV-1 vector, wherein the genome of the modified HSV-1 vector comprises an inactivated deletion of the US8 coding sequence and / or non-coding region; and wherein the modified HSV-1 vector is capable of growing in a suspension cell line.

2. The modified HSV-1 vector according to claim 1, wherein the inactivated deletion comprises a single nucleotide substitution in the US8 coding sequence.

3. The modified HSV-1 vector according to claim 1 or 2, wherein the parental HSV-1 vector is the human HSV-1F strain.

4. The modified HSV-1 vector according to claim 3, wherein the single nucleotide substitution is located at nucleotide position 348 of the US8 coding sequence, resulting in a W116STOP mutation, and this nucleotide position is numbered relative to the position in the wild-type US8 gene (SEQ ID NO: 3).

5. The modified HSV-1 vector according to claim 2, wherein the substituted US8 coding sequence comprises the nucleic acid sequence of SEQ ID NO:

4.

6. The modified HSV-1 vector according to any one of the preceding claims, wherein the modified HSV-1 vector is a replication-competent HSV-1 vector, a defective helper virus-independent HSV-1 vector, a helper HSV-1 vector or an HSV-1 amplicon vector.

7. The modified HSV-1 vector according to claim 6, wherein the modified HSV-1 vector is an HSV-1 amplicon vector.

8. The HSV-1 amplicon vector according to claim 7, wherein the modified HSV-1 amplicon vector comprises a mutant US8 protein expressed by SEQ ID NO: 4, and the mutant US8 protein comprises a W116STOP mutation relative to the wild-type US8 protein.

9. The HSV-1 amplicon vector according to any one of the preceding claims, wherein the HSV-1 amplicon vector comprises a non-functional US8 protein or lacks the US8 protein.

10. The modified HSV-1 vector according to any one of the preceding claims, which comprises a genome containing an exogenous expression cassette.

11. The modified HSV-1 vector according to claim 10, wherein the expression cassette comprises at least one nucleic acid sequence encoding a gene product.

12. A pharmaceutical composition comprising the modified HSV-1 vector according to any one of claims 1-11 and a pharmaceutical excipient.

13. A kit comprising the modified HSV-1 vector according to any one of claims 1-11 and an instruction manual.

14. A method for producing an HSV-1 vector in a suspension cell line, the method comprising: Infecting a suspension cell line with the modified HSV-1 vector according to any one of claims 1-11; and culturing the infected cells.

15. The method according to claim 14, wherein the suspension cell line is selected from CHO, HeLa, H-9, Jurkat, C6 / 36, High Five, S2, Sf21, Sf9, PC-1 and HEK293.

16. The method according to claim 15, wherein the suspension cell line is HEK293.

17. A method for producing HSV-1 amplicon vectors in a suspension cell line, wherein the method comprises: Infecting a suspension cell line with a helper virus-dependent packaging system comprising: an HSV-1 amplicon vector or an HSV-1 amplicon plasmid, and a helper HSV-1 vector; and wherein the helper HSV-1 vector comprises a US8 gene containing the nucleic acid sequence of SEQ ID NO:4 or an inactivated deletion of the US8 gene; and culturing the infected cells.

18. The method according to claim 17, wherein the suspension cell line is selected from CHO, HeLa, H-9, Jurkat, C6 / 36, High Five, S2, Sf21, Sf9, PC-1, and HEK293.

19. The method according to claim 18, wherein the suspension cell line is HEK293.

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