Compositions and methods for gene expression in the brain

Abstract

The present invention provides a method for efficiently delivering a transgene to the brain of a subject. The method includes intrathecal administration or one or more injections into the striatum of a replication defective herpes simplex virus (rdHSV) vector comprising an inactivating mutation of one copy of infected cell protein 0 (ICP0) HSV gene; and wherein the rdHSV-1 vector expresses a functional ICP0 protein from the other copy of the ICP0 gene.

Description

[0001] DESCRIPTION

[0002] TITLE: Compositions and Methods for Gene Expression in the Brain

[0003] FIELD OF THE APPLICATION

[0004] This application relates to methods and compositions for delivering a transgene to the brain of the subject.

[0005] BACKGROUND

[0006] Wild type HSV-1 does not cross the BBB. However, many studies have shown that it can be efficiently administered intrathecally, both in the brain and in the spinal cord. However, studies have demonstrated that various defective HSV vectors do not efficiently propagate to and / or express in different compartments of the brain. If a HSV vector could propagate well into different segments of the brain then a series of pathologies of the CNS could be targets for gene therapy or gene editing approaches, via intra-cerebral or intrathecal administration. Thus, there is a need for an HSV vector that could propagate well into different segments of the brain.

[0007] SUMMARY OF THE INVENTION

[0008] This application provides a method of delivering a transgene to the brain of a subject, the method comprising administering to the subject a pharmaceutical composition comprising a replication-defective herpes simplex type 1 virus (HSV-1) comprising an inactivating mutation of one copy of infected cell protein 0 (ICPO) HSV gene while expressing the other copy of ICPO and one or more polynucleotides encoding the transgene; and a pharmaceutically acceptable carrier; wherein the pharmaceutical composition is administered intrathecally or by one or more injections into the striatum of the subject. The method of the present invention allows for efficient transgene delivery to one or more neurons of the brain. The method also allows for fewer injections and / or a lower number of viral particles to provide efficient transgene delivery.

[0009] In some embodiments, the viral vector used in the method of this application is a herpes simplex virus (HSV) vector, preferably a defective viral vector derived from HSV, such as a replication defective HSV vector. Preferably the HSV vector is an HSV-1 vector or an HSV-2 vector, more preferably a defective viral vector derived from HSV-1 or HSV-2, such as a replication defective HSV-1 vector or a replication defective HSV-2 vector. Preferably the HSV vector is as a replication defective HSV-1 vector.

[0010] In embodiments, the HSV vector comprises an expression cassette inserted therein. In a preferable embodiment, the viral vector used in the method of this application is a defective viral vector derived from HSV-1, wherein the expression cassette is inserted in the LAT (Latency Associated Transcripts) locus in the defective viral vector derived from HSV-1. In some embodiments, the viral vector comprises a promoter for driving the long-term expression of a transgene.

[0011] In some embodiments, the viral vector of the invention is administered directly into the intrathecal space of the subject. In some embodiments, the viral vector of the invention is administered via one or more injections into the striatum. In embodiments, the viral vector of the invention is stereotaxically injected into the striatum.

[0012] Also provided in this application is a method of efficiently delivering a transgene to the brain of a subject comprising administrating to the subject a therapeutically effective amount of a replication defective viral vector as described herein comprising a transgene.

[0013] Preferably, in any of the methods described herein, the transgene is expressed in the in the striatum, the substantia nigra, subthalamic nucleus, ependymal cells of the lateral ventricle, and the cortex of the brain, following injection in the striatum. Preferably, in any of the methods described herein, the transgene is further expressed in the pre-frontal cortex following injection in the striatum. Expression in the neural cells of the brain demonstrates axonal transport of the replication defective viral vector of the invention following administration.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1A, Fig IB and Fig. 1C demonstrate that transgene expression is visible at 3 days and 10 days post injection at injection site and also in substantia nigra and cortex demonstrating axonal transport in CNS.

[0016] Fig. 2 demonstrates localization of transgene expression in neural cells.

[0017] Fig. 3 demonstrates that the defective viral vector of the invention efficiently targets projecting neurons of the substantia nigra and cortex 3 days following administration into the striatum. Fig. 4 depicts colocalization of mGreen lantern with tyrosine hydroxylase (TH) staining, which demonstrates that the HSV1 vector transduced dopaminergic neurons of the substantia nigra after intra-striatal administration.

[0018] Fig. 5 shows that a vector according to the invention transduced cortical neurons projecting in striatum from the contralateral hemisphere.

[0019] Fig. 6A through Fig. 6C depict stable mGreen lantern expression following retrograde transport of the vector. Fig. 6A shows that the number of cortical neurons expressing mGreen lantern was similar between 1 and 6 weeks. Fig. 6B shows the number of vector episomes in the cortex was stable between 1 and 6 weeks as determined by ddPCR. Fig. 6C depicts that mGreen lantern expression (number of mGreen lantern transcripts (RNA)) in the cortex was stable between 1 and 6 weeks as determined by reverse transcription-ddPCR. Mean + / - SD. Each dot represents one injected hemisphere.

[0020] Fig. 7 depicts mGreen lantern expression was seen at similar levels in the cortex after intra-striatal injection of both HSV-1A and HSV-1B vectors, demonstrating that both EFla and CAG promoters drove mGreen lantern expression in cortical neurons.

[0021] Fig. 8A and Fig. 8B depicts vector maps for the HSV-1 A (Fig. 8A) and HSV-1B (Fig. 8B) vectors from the examples.

[0022] DETAILED DESCRIPTION

[0023] 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, references to “the method” includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0024] The term “comprising”, which is used interchangeably with “including”, “containing”, or “characterized by”, is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps.

[0025] The phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the invention compositions and methods corresponding to the scope of each of these phrases. Thus, a composition or method comprising recited elements or steps contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0026] Unless defined otherwise, 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 in the practice or testing of the invention, the preferred methods and materials are now described.

[0027] The term “subject” as used herein refers to any individual or patient to which the subject methods are performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may 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.

[0028] “Drug tolerance” as used herein is meant to describe a subject’s reduced reaction to a drug, usually following the repeated use of the drug. Increasing the drug’s dosage may reamplify the drug's effects; however, this may accelerate tolerance, further reducing the drug's effects. In some embodiments, the terms “tolerance”, “resistance”, and “insensitivity” may be used interchangeably to describe the reduction in effectiveness of a medication.

[0029] A “therapeutic effect,” as used herein, encompasses a therapeutic benefit and / or a prophylactic benefit as described herein.

[0030] As used herein, the terms “reduce” and “inhibit” are used together because it is recognized that, in some cases, a decrease can be reduced below the level of detection of a particular assay. As such, it may not always be clear whether the expression level or activity is “reduced” below a level of detection of an assay, or is completely “inhibited.” Nevertheless, it will be clearly determinable, following a treatment according to the present methods.

[0031] As used herein, the term “reduce” also refers to lesser number of an activity, such as the total number of injections required to achieve the desired result.

[0032] As used herein, “treatment” or “treating” means to administer a composition to a subject or a system with an undesired condition. The condition can include a disease or disorder. “Prevention” or “preventing” means to administer a composition to a subject or a system at risk for the condition. The condition can include a predisposition to a disease or disorder. The effect of the administration of the composition to the subject (either treating and / or preventing) can be, but is not limited to, the cessation of one or more symptoms of the condition, a reduction or prevention of one or more symptoms of the condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or minimization of the chances that a particular event or characteristic will occur.

[0033] As used herein, an “inactivating mutation” of a gene is any complete or partial deletion of the gene or any substitution or addition to the gene that prevents expression of a functional protein. An “inactivating mutation” includes any mutation that results in a gene or regulon product (RNA or protein) having reduced, undetectable, or eliminated quantity and / or function (e.g., as compared to a corresponding sequence lacking the inactivating mutation). Examples of inactivating mutations may include, but are not limited to, deletions, insertions, point mutations, and rearrangements in transcriptional control sequences (promoters, enhancers, insulators, etc.) and / or coding sequences of a given gene or regulon. Any suitable method of measuring the quantity of a gene or regulon product known in the art may be used, including, for example, qPCR, Northern blots, RNAseq, western blots, ELISAs, etc.

[0034] As used herein, the “cortex” of the brain, also known as the cerebral cortex, is the outer layer of neural tissue of the cerebrum of the brain in humans and other mammals. The cerebral cortex mostly consists of the six-layered neocortex, with just 10% consisting of the allocortex. It is separated into two cortices, by the longitudinal fissure that divides the cerebrum into the left and right cerebral hemispheres. The two hemispheres are joined beneath the cortex by the corpus callosum. The cerebral cortex is the largest site of neural integration in the central nervous system and plays a key role in attention, perception, awareness, thought, memory, language, and consciousness.

[0035] As used herein, the striatum (also called the striate nucleus) is a cluster of neurons in the subcortical basal ganglia of the forebrain.

[0036] As used herein, stereotaxically refers to the precise directing the tip of a delicate instrument (such as a needle) or beam of radiation in three planes using coordinates provided by medical imaging in order to reach a specific locus in the body.

[0037] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0038] 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 a 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%.

[0039] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, a-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0040] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0041] As used herein, a “regulatory gene” or “regulatory sequence” is a nucleic acid sequence that encodes products (e.g., transcription factors) that control the expression of other genes.

[0042] As used herein, a “protein coding sequence” or a sequence that encodes a particular protein or polypeptide, is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' terminus (N-terminus) and a translation stop nonsense codon at the 3' terminus (C-terminus). A coding sequence can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence will usually be located 3' to the coding sequence.

[0043] As used herein, the term “expression cassette” or “transcription cassette” refers to a distinct component of vector DNA consisting of a gene and regulatory sequence to be expressed by a transfected cell. In each successful transfection, the expression cassette directs the cell’s machinery to make RNA and protein(s). Some expression cassettes are designed for modular cloning of protein-encoding sequences so that the same cassette can easily be altered to make different proteins. An expression cassette can be composed of one or more genes and the sequences controlling their expression. An expression cassette comprises at least three components: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site.

[0044] As used herein, the qualifier “essential” in the expressions “essential genes” or “non- essential genes”, means that the given gene is essential (or not) for achieving multiplication and packaging of the virus genome, thus generating infectious progeny virus particles. HSV-1 essential genes include ULI, UL5-UL9, UL12, UL14, UL15, UL17-UL19, UL22, UL25-UL38, UL42, UL48, UL49, UL52-UL54, US6, ICP4 (2 copies). HSV-1 non-essential genes include ICP34.5 (2 copies), ICPO (2 copies), LAT (2 copies), UL2-UL4, UL10, UL11, UL13, UL16, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43-UL47, UL50, UL51, UL55, UL56, US1- US5, US7-US12.

[0045] 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 directing RNA polymerase to bind to DNA and initiating RNA synthesis. A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state), it may be an inducible promoter (i.e., a promoter whose state, active / “ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular compound or protein), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.; e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process). For purposes of the present invention, a promoter sequence includes at least the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as RNA polymerase binding domains. Eukaryotic promoters will often, but not always, contain "TATA" boxes and other DNA motifs, such as "CAT" or "SP1" boxes.

[0046] As used herein, the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene. A “gene” may also include non- translated sequences located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non- translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0047] As used herein, the terms “functionally linked” and “operably linked” are used interchangeably and refer to a functional relationship between two or more DNA segments, in particular gene sequences to be expressed and those sequences controlling their 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 modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Promoter regulatory sequences that are operably linked to the transcribed gene sequence are physically contiguous to the transcribed sequence.

[0048] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0049] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0050] The following eight groups each contain amino acids that are conservative substitutions for one another:

[0051] 1) Alanine (A), Glycine (G);

[0052] 2) Aspartic acid (D), Glutamic acid (E);

[0053] 3) Asparagine (N), Glutamine (Q);

[0054] 4) Arginine (R), Lysine (K);

[0055] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0056] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0057] 7) Serine (S), Threonine (T); and

[0058] 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0059] A conservative substitution (also called conservative replacement or conservative mutation) may include substitution such as basic for basic, acidic for acidic, polar for polar, etc. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets 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.

[0060] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0061] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The invention provides polypeptides that are substantially identical to the polypeptides, respectively, exemplified herein, as well as uses thereof including, but not limited to, use for treating or preventing neurological diseases or disorders, e.g., neurodegenerative diseases or disorders, and / or treating SCI. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or the entire length of the reference sequence.

[0062] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0063] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 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 alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 1970, 2:482c, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 1970, 48:443, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA, 1988, 85:2444, by 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, supplement).

[0064] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are 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. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word 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 searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, 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) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0065] 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 smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest 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.

[0066] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). In various embodiments, nucleic acids are isolated when purified away from other cellular components or other contaminants (e.g., other nucleic acids or proteins present in the cell) by standard techniques including, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well-known in the art. See e.g., F. Ausubel, et al., ed., Current Protocols in Molecular Biology, 1987, Greene Publishing and Wiley Interscience, New York. In various embodiments, a nucleic acid is, for example, DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0067] As used herein “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.

[0068] As used herein, the term “neuron” includes a neuron and a portion or portions thereof (e.g., the neuron cell body, an axon, or a dendrite). The term “neuron” as used herein denotes nervous system cells that include a central cell body or soma, and two types of extensions or projections: dendrites, by which, in general, the majority of neuronal signals are conveyed to the cell body, and axons, by which, in general, the majority of neuronal signals are conveyed from the cell body to effector cells, such as target neurons or muscle. Neurons can convey information from tissues and organs into the central nervous system (afferent or sensory neurons) and transmit signals from the central nervous systems to effector cells (efferent or motor neurons). Other neurons, designated interneurons, connect neurons within the central nervous system (the brain and spinal column). Certain specific examples of neuron types that may be subject to treatment or methods according to the invention include cerebellar granule neurons, dorsal root ganglion neurons, and cortical neurons.

[0069] The term “neuronal degeneration” is used broadly and refers to any pathological changes in neuronal cells, including, without limitation, death or loss of neuronal cells, any changes that precede cell death, and any reduction or loss of an activity or a function of the neuronal cells. The pathological changes may be spontaneous or may be induced by any event and include, for example, pathological changes associated with apoptosis. The neurons may be any neurons, including without limitation sensory, sympathetic, parasympathetic, or enteric, e.g., dorsal root ganglia neurons, motor neurons, and central neurons, e.g., neurons from the spinal cord, including inter-neurons. Neuronal degeneration or cell loss is a characteristic of a variety of neurological diseases or disorders, e.g., neurodegenerative diseases or disorders. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a motor neuron. In some embodiments, the neuron is a damaged spinal cord.

[0070] As used herein, the term “afferent neurons” carry information from sensory receptors of the skin and other organs to the central nervous system (i.e., brain and spinal cord), whereas “efferent neurons” carry motor information away from the central nervous system to the muscles and glands of the body. “Afferent” in this application is referred to carrying inward to a central organ or section, as nerves that conduct impulses from the periphery of the body to the brain or spinal cord.

[0071] As used herein, the term “systemic” is referred to pertaining to or affecting the whole body rather than one part of it. The term “region-specific” as used herein is referred to as only affecting a particular region (such as a particular cell type, a particular dermatome, and a particular spinal nerve) of the body and having zero or minimal impact on the rest of the body.

[0072] The present invention provides a gene therapy capable of delivering a transgene coding for a therapeutic protein or upregulating or downregulating one or more genes may provide a therapeutic approach in the brain, wherein a viral vector is used for the delivery of a therapeutic gene product or an active segment thereof. In embodiments, expression of the transgene can be detected only in regions of the brain that are either at the point of injection or regions connected thereto by axonic junction.

[0073] In one aspect, the invention provides a method of delivering a transgene to the brain of a subject, the method comprising administering to the subject a pharmaceutical composition comprising a replication-defective herpes simplex virus (HSV) comprising an inactivating mutation of one copy of infected cell protein 0 (ICPO) HSV gene while expressing the other copy of ICPO and one or more polynucleotides encoding the transgene; and a pharmaceutically acceptable carrier; wherein the pharmaceutical composition is administered intrathecally or by one or more injections into the striatum of the subject. The method of the present invention allows for efficient transgene delivery to neurons of the brain with the need for fewer injections. Preferably, the replication-defective HSV vector is a replication-defective herpes simplex type 1 virus (HSV-1) vector.

[0074] In other words, the replication-defective herpes simplex virus of the invention (also referred to herein as “rdHSV” or “rdHSV-1”), does not allow for the expression of one copy of IPCO while preserving the expression of the other copy of ICPO. This results in the rdHSV vector of the invention providing a functional copy of the ICPO protein from the preserved copy of the ICPO gene.

[0075] Preferably, in any of the methods described herein, the transgene is expressed in the striatum, the substantia nigra, subthalamic nucleus, ependymal cells of the lateral ventricle, and the cortex of the brain, following injection in the striatum. Preferably, in any of the methods described herein, the transgene is further expressed in the pre-frontal cortex following injection in the striatum. Expression in the neural cells of the brain demonstrates axonal transport of the replication defective viral vector of the invention following administration.

[0076] Herpes Simplex Virus (HSV)

[0077] The term “viral vector” or “viral expression vector” as used herein refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle. In the context of the present invention, the term “viral vector” has to be understood broadly as including nucleic acid vector (e.g., DNA viral vector) as well as viral particles generated thereof. In this application, the viral expression vector is an adeno- associated virus (AAV) vector or a herpes simplex virus (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, even more preferably a replication-defective viral vector derived from HSV-1. As used herein the term “replication-defective viral vector” shall refer to viral vectors that are missing genes or parts of genes necessary to complete successfully the viral life cycle in order to replicate.

[0078] The term "AAV" refers to the Adeno- Associated Virus itself or to derivatives thereof including recombinant AAV vector particles. Furthermore, as used herein, the term "AAV" includes many different serotypes, which have been isolated from both human and non-human primate samples. Preferred AAV serotypes are the human serotypes, more preferably human AAV of serotypes 2, 5 and 9, most preferably human AAV of serotype 5, which is the serotype displaying the highest level of neurotropism.

[0079] The term “herpes simplex virus (HSV)” is a complex, non-integrating DNA virus capable of infecting a very wide range of human and animal cells. HSV encompasses two serotypes, herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). The genome of HSV-1 has a size of approximately 153-kbp. It contains some 90 protein-encoding genes and more than 12 microRNA. The HSV-1 genome is composed of two unique segments, UL and US, each flanked by inverted repeats that encode critical diploid genes.

[0080] Importantly, the HSV genome comprises 2 copies of infected cell protein 0 (ICPO). Each copy is located in the repeated region of the HSV genome, one copy in the Terminal Repeat Long region, and the other copy in the Internal Repeat Long region. ICPO is an immediate early protein, expressed under the control of a promoter activated by VP16, a HSV protein. ICPO is a critical viral protein that activates expression of HSV genes or transgenes by disrupting inhibitory cellular functions able to block the viral cycle at different levels. ICPO can inhibit or target the destruction of cellular proteins involved in the innate immune responses and is also able to disrupt the repressive forces brought about by the heterochromatinization of the virus genome and by ND 10 bodies. On the other hand, in wild type HSV, ICPO has been shown to be toxic for proliferating cells (for example for glial cells).

[0081] The terms "replication defective HSV", “defective HSV vector”, or “defective viral vector”, are used interchangeably herein, to describe a helper-independent vector comprising an inactivating mutation for 1 copy of the gene coding for the essential protein ICPO while expressing the other copy of ICPO. In some embodiments, the one copy of the ICPO gene comprising the inactivating mutation is completely deleted in the defective HSV vector. In a preferred embodiment, the inactivating mutation of the one copy of the IPCO gene is an inactivating mutation in the copy of the ICPO gene present among the LAT, ICPO, UL34.5 cluster from the IRL (Internal Repeat Long) region of the HSV vector. Preferably, the replication-defective vector of the invention expresses the other copy of ICPO under the wildtype promoter.

[0082] Without wishing to be bound to any particular theory, it is believed that the deletion of both copies of ICPO can fully eliminate its cytotoxicity but, in doing so, significantly weakens the ability of the virus to counteract the antiviral cellular functions, eventually ending in downregulation or shutoff of transgene expression. Keeping one copy of ICPO is believed to reduce its cytotoxic effects while still allowing the virus to efficiently counteract cellular antiviral functions and viral genome repression, thereby resulting in sustained transgene expression.

[0083] In a preferred embodiment, the replication-defective vector further comprises inactivating mutations for the genes coding for the essential proteins ICP4 and ICP27. The ICP4 gene is present in two copies, located in the inverted repeated sequences known as c and c' of the virus genome. The gene encoding ICP27 is located in the unique long (UL) sequence of the virus genome. Preferably, both copies of the ICP4 comprise an inactivating mutation. Preferably, both copies of the ICP4 gene are completely deleted. Preferably, the ICP27 gene is completely deleted. Preferably, replication-defective vectors according to the invention can comprise additional inactivating mutations in genes encoding non-essential proteins such as ICP22, ICP34.5 (1 copy), UL55, UL56, and UL41 proteins.

[0084] Preferably, replication-defective vectors according to the invention comprise inactivating mutations in genes encoding essential proteins ICP4 (both copies), ICP27, ICPO (1 copy) and non-essential proteins ICP22, ICP47, and ICP34.5 (1 copy).

[0085] It is contemplated that a transgene, with or without extraneous control elements, can be inserted into the HSV vector according to the invention. Preferably, the HSV vector comprising the transgene is capable of persistent expression (also referred to as “long term expression”) of the transgene. Preferentially, replication-defective vectors according to the present invention carry a transcription cassette(s) (also referred to as an “expression cassette”) embedded into the LAT (Latency Associated Transcripts) locus which is a repeated locus that is contained in the inverted repeated sequences known as b and b' of the virus genome. (See Berthomme et al., “Evidence for bidirectional element located downstream from the herpes virus simplex type 1 latency-associated promoter that increases its activity during latency”, JOURNAL OF VIROLOGY, 2000, 74, 3613 - 3622; and Berthomme et al. “Enhancer and long-term expression functions of herpes simplex virus type 1 latency-associated promoter are both located in the same region”, JOURNAL OF VIROLOGY, 2001, 75, 4386 - 4393; the contents of which are incorporated by reference.)

[0086] In a preferable embodiment, the HSV vector includes a transgene inserted in operable connection with one or more LTEs and / or DNA insulator sequences within the HSV vector genome. By "operably connected," it is to be understood that the one or more LTEs and / or DNA insulator sequences permit the transgene to be expressed in a cellular environment in which genetic elements (i.e., "genes") otherwise present within the HSV genome are transcriptionally silent.

[0087] In some embodiments, the modified HSV vector may further comprise a DNA sequence, for example an endogenous and / or foreign DNA sequence encoding a cell targeting protein, introduced into a genic region within the genome. The cell targeting protein can retarget virus entry into any tissue or cell type of interest. Cell targeting genes for insertion in HSV-1 vector used in the invention can include, but are not limited to, HER-2, IL13a2 or modified versions thereof. Examples of HSV modified glycoprotein D can be found, for example, in EP3469071, which is incorporated herein by reference.

[0088] These defective HSV vectors are multiplied in cell lines expressing simultaneously the proteins ICP4 and ICP27 (Marconi et al, “HSV- 1- derived helper-independent defective vectors, replicating vectors and amplicon vectors, for the treatment of brain diseases”, CURRENT OPINION IN DRUG DISCOVERY AND DEVELOPMENT, 13, 2010, 169 - 183; the contexts of which are incorporated by reference).

[0089] W02006 / 050211 discloses the use of a defective HSV-1 vector for gene therapy for treating pain. However, the vectors according to the invention differ from the vector described in W02006 / 050211 in several significant respects, which are important in regard to the usefulness and efficacy of the vectors according to the invention. Most important, transgenic transcription cassettes according to the invention are introduced into the LAT locus, as this region contains both the LTE and the DNA insulator sequences (INS) that confer long-term expression to the promoters driving transgene expression in transcription cassettes according to the invention, whereas the vector described in W02006 / 050211 was conceived and proved for short-term action and, therefore, their transcription cassettes were not introduced into the LAT regions.

[0090] The expression "recombinant DNA" as used herein describes a nucleic acid molecule, i.e., a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, 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 virus means a virus carrying a recombinant genome or a genome that has been manipulated to introduce mutations, deletions or one or more heterologous polynucleotides, including genes. The term "recombinant" as used with respect to a protein or polypeptide, means a polypeptide produced by expression of a recombinant nuclei acid. The term "recombinant" as used with respect to a host cell means a recombinant vector that carries recombinant DNA within the host cell or a cell that contains recombinant DNA inserted in its genome. The term " infection" refers to the ability of a viral vector to enter a host cell or organ or subject, or the ability of a gene product of the viral vector to enter a host cell.

[0091] Injection or intrathecal administration of replication defective HSV vectors of the invention allow for infection of neighboring neurons and establish latent infections in the nucleus of these neurons. For example, following administration, replication defective vectors derived from HSV-1 as disclosed herein will reach the connected neurons, either by directly infecting the cell bodies, or after travel along the nerve axon in a retrograde direction, where they will stably express the transgene, provided that adequate promoters drive their long-term expression and that adequate DNA insulators protect the region from heterochromatinization. In some embodiments, the promoters may comprise both neuron-specific promoters and ubiquitous promoters. Preferably, expression of the transgene is found in the cortex following administration of the replication-defective HSV vector of the invention in the striatum.

[0092] Preferably, the replication defective HSV vector is a replication defective HSV-1 vector.

[0093] Pre-HSV-1 Vector

[0094] In some embodiments, the replication defective HSV viral vector of the invention and as used in the methods herein may be produced starting with a pre-HSV-1 vector.

[0095] As used herein, a “pre-HSV-1 vector” is a mini-HSV-1 backbone (aka “mini-HSV-1” or “a pre-HSV-1 vector”), wherein non-essential genes, essential genes, or combinations thereof have been deleted to arrive at a genome comprising less than 130 kbp and greater than 75 kbp. Pre-vectors or mini vector "backbones" embodying the invention are described with the understanding that, as "backbones," it is contemplated that a transgene, with or without extraneous control elements, can be inserted therein.

[0096] In some embodiments, clusters of genes that could be deleted include, but are not limited to, genes UL2, UL3, UL4 (10.200 - 12.600); genes UL10, ULI 1 (23.200 - 25.200); gene UL16 (30.200 - 31.400); genes UL20, UL21 (40.800 - 43.700); genes UL23, UL24 (46.700 - 48.600); genes UL39, UL40, UL41 (86.400 -92.700); genes UL43 to UL47 (94.700 - 103.200); genes UL50, UL51 (107.700 - 109.100); genes UL55, UL56 (115.400 - 117.100); one copy of genes LAT, ICP0, UL34.5 (IRL) (118.700 - 126.100); genes US2 to US5 (134.000 - 138.200); and / or genes US7 to US12 (139.700 - 145.600).

[0097] In some embodiments, the mini-HSV-1 comprises a genome wherein at least 30 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein at least 40 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein at least 45 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein at least 50 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein at least 55 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein at least 60 kbp have been deleted. In some embodiments, the mini- HSV-1 comprises a genome wherein at least 65 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein at least 75 kbp have been deleted.

[0098] In some embodiments, the mini-HSV-1 comprises a genome wherein 25 kbp to 80 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein 30 kbp to 75 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein 35 kbp to 70 kbp have been deleted. In some embodiments, the mini-HSV-1 comprises a genome wherein 40 kbp to 60 kbp have been deleted.

[0099] A pre-HSV-1, wherein non-essential genes, essential genes, or combinations thereof have been deleted to arrive at a genome comprising less than 130 kbp and greater than 75 kbp, can allow a transgene, with or without extraneous control elements, to be inserted therein. As a nonlimiting example, a recombination technique based on the markerless Red recombination system may be used to generate the scarless point mutations, deletions, and insertion of smaller and larger sequences in the recombinant viral vectors used in this invention (e.g., Tischer et al., En Passant Mutagenesis: A Two Step Markerless Red Recombination System. Chapter 30, In Vitro Mutagenesis Protocols: Third Edition, Methods in Molecular Biology, vol. 634. DOI 10.1007 / 978-l-60761-652-8_30, © Springer Science+Business Media, LLC 2010). In some embodiments, as used in this invention, a pre-HSV-1 vector comprises a modified HSV-1 genome wherein non-essential genes, essential genes, or combinations thereof have been deleted to arrive at a genome comprising less than 130 kbp and greater than 75 kbp.

[0100] In some embodiments, one or more exogenous genes of interest is introduced into the pre-HSV-1 vector. In some embodiments, a combination of one or more HSV-1 essential genes, one or more HSV-1 non-essential genes, and one or more exogenous genes of interest is introduced into the pre-HSV-1 vector. In some embodiments, a combination of one or more HSV-1 essential genes and one or more exogenous genes of interest is introduced into the pre- HSV-1 vector. In some embodiments, a combination of one or more HSV-1 non-essential genes and one or more exogenous genes of interest is introduced into the pre-HSV-1 vector.

[0101] It is important that the pre-HSV-1 vectors used herein maintain enough of the HSV-1 genome so as to not become HSV-1 amplicons. By "Amplicon or amplicon vector" it is meant a helper-dependent vector, the genome of which lacks most or all HSV genes coding for virus proteins. The genome of amplicon vectors is a concatemeric DNA composed of multiple copies in tandem of a plasmid -known as the amplicon plasmid- that carries one origin of DNA replication and one packaging signal from HSV-1 genome. In cells expressing the full set of structural, replication and DNA packaging functions from HSV-1, resulting from the presence of an HSV-1 genome acting as helper, the amplicon plasmid is amplified by a rolling-circle mechanism into long head-to-tail concatemers that are then cleaved and packaged, up to one genome size, into HSV-1 virions (Kwong and Frenkel, 1985; Bataille and Epstein, 1997). Amplicon vectors are thus a concatemeric plasmidic DNA packaged into HSV-1 particles.

[0102] In contrast to amplicons, the pre-HSV-1 vectors of the invention are a helper-independent vector platform, which means that they do not need the presence of an HSV-1 genome acting as a helper virus for vector replication and packaging.

[0103] Transgene

[0104] As used herein, the term "transgene" refers to a polynucleotide that is capable of being transcribed into RNA and translated and / or expressed under appropriate conditions, after being introduced into a cell. In some aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. The transgene can comprise one or more nucleic acid sequence (i.e., one or more polynucleotides) encoding one or more RNAs and / or one or more polypeptides or portions of one or more polypeptides to be expressed in a cell into which the nucleic acid sequence is introduced.

[0105] The term "transgene" includes (1) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been introduced; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been introduced. By "mutant form" is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that the transgene product will be secreted from the cell, or the transgene may include both a leader peptide or signal sequence plus a membrane anchor peptide, or even be a fusion protein between two naturally occurring proteins or part of them, such that the transgene will remain anchored to cell membranes, or a sequence that allows the protein to accumulate in a specific region of the cell, such as a nuclear localizing signal.

[0106] In some embodiments, the vector comprises two transgenes, wherein each transgene encodes the same or different polypeptide (e.g., protein) to be expressed. In some embodiments, the vector comprises at least a first transgene and a second transgene, wherein the first transgene and the second transgene are the same or different. In embodiments, the first and second transgene are the same. In embodiments, the first and second transgene are the same.

[0107] In some embodiments, the vector comprises a transgene that is polycistronic. In some embodiments, the polycistronic transgene encodes first transgene on a first open reading frame (ORF) and a second transgene on a second open reading frame (ORF).

[0108] As used herein, the transgene can include, but are not limited to, a reporter gene (e.g., GFP, RFP, luciferase, or fused protein, etc.) driven by a transient promoter serving as internal expression control or for biodistribution studies; recombinases driven by an inducible promoter to allow in vivo modifying cellular or viral genes; or combinations thereof. The use of reporter genes such as, but not limited to, cherry, RFP, GFP, or CFP, can facilitate the identification of the recombined genome and to score both infectious particles (PFU) and transducing units (TU).

[0109] Transcription cassette

[0110] The transgene can be part of a transcription cassette (also referred to herein as an “expression cassette”), wherein the transgene is operably linked to a promoter, which drives the expression of the transgene. The transcription cassette can further comprise a polyadenylation signal or any enhancer as known in the art.

[0111] Long term expression.

[0112] By "long-term expression sequence" or "long-term expression element (LTE)" it is meant a nucleotide sequence that when operably linked to a foreign DNA of interest (i.e., a transgene or an expression cassette comprising the transgene) allows for sustained expression of a gene product for more than 15 to 45 days or 30 to 45 days, or from 45 to 90 days, or from 90 to 365 days, or 365 days to several years or even during the life of the patient. Long-term expression (LTE) sequences were identified in HSV-1 as a region of the latency- associated transcripts (LAT), which originate from the LAT-associated promoter (LAP). This LTE is located downstream of the LAT transcription start site.

[0113] In some embodiments, transgene of interest can be introduced into a LAT (Latency Associated Transcripts) locus, which is a repeated locus that is contained in the inverted repeated sequences known as b and b' of the virus genome. The b and b' sequences of the virus genome are also known as TRL (Terminal Repeat Long) and IRL (Internal Repeat Long), respectively. The c' and c sequences are also known as IRS (Internal Repeat S) and TRS (Terminal Repeat S), where L and S refer respectively to the unique long (L) and unique short (S) sequences of the HSV-1 genome.

[0114] In some embodiments, the virus genome contains both LAT regions, one in the TRL and the other in the IRL. In some embodiments one of the LAT regions, either in the TRL or in the IRL has been deleted. In some embodiments, when the vector genome contains the two LAT loci, the transgene of interest can be introduced into both loci, in the TRL region and in the IRL region. In some embodiments, when the LAT loci in the IRL region is deleted, the transgene of interest can be introduced into the LAT locus in the TRL region only. In some embodiments, when the LAT loci in the TRL region is deleted, the transgene of interest can be introduced into the LAT locus in the IRL region only.

[0115] The LAT locus includes an upstream DNA insulator (INS) sequence, the Latency Associated Promoter (LAP), a region conferring Long-Term Expression (LTE) and a downstream DNA insulator (INS). In some embodiments, the transgene of interest is introduced either between the Latency Associated Promoter (LAP) and the Long-Term Expression (LTE) region, or between the LTE region and the DNA insulator (INS) sequence present downstream of the LTE.

[0116] In embodiments, the transgene is part of a transcription cassette (also referred to as an expression cassette) Preferably, the transcription cassette is placed either 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 present downstream of the LTE (site 2). Replication defective HSV-1 vectors according to the present invention carry transcription cassette(s) expressing a transgene of interest or an active fragment thereof driven by a promoter. Importantly, the LAT locus contains both the LTE and the DNA insulator sequences (INS) that confer long-term expression to a polynucleotide encoding a transgene introduced into this site.

[0117] Indeed, viruses harboring a DNA fragment 3' of the LAT promoter maintained detectable promoter expression throughout latency (Lokensgard et al, “The latency-associated promoter of herpes simplex virus type 1 requires a region downstream of the transcription start site for longterm expression during latency”, Journal of Virology, 1997, 71, 6714-6719; Berthomme et al., “Evidence for bidirectional element located downstream from the herpes virus simplex type 1 latency-associated promoter that increases its activity during latency”, JOURNAL OF VIROLOGY, 74, 2000, 3613 - 3622; and Berthomme et al. “Enhancer and long-term expression functions of herpes simplex virus type 1 latency-associated promoter are both located in the same region”, JOURNAL OF VIROLOGY, 75, 2001, 4386 - 4393; the contents of which are incorporated by reference.) Preferably, the LTE is comprised between about 1.5 kb to about 3 kb downstream of the LAT transcription start site (Perng et al., “The spontaneous reactivation function of the herpes simplex virus type 1 LAT gene resides completely within the first 1.5 kilobases of the 8.3-kilobase primary transcript”, JOURNAL OF VIROLOGY, 1996, 70, 976 - 984; the contents of which are incorporated by reference.) More recently, additional sequences, known as DNA insulators, have also been described both upstream and downstream the LTE region (Amelio et al., “A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcription region binds CCCTC-binding factor and displays enhancerblocking and silencing activities”, JOURNAL OF VIROLOGY, 2006, 80, 2358 - 2368; the contents of which are incorporated by reference). These sequences also contribute to provide long-term expression to a given transcription cassette probably by inhibiting epigenetic silencing and are incorporated in the present invention as part of the LTE elements, to confer long-term expression to the expression cassette. Interestingly, sequences conferring long-term expression to the transcription cassette (both the LTE and the DNA insulator sequences) can be placed either upstream and / or downstream the expression cassette.

[0118] Those of skill in the art will recognize that other LTE-like sequences, as well as other DNA insulator sequences, have been described and are continually being discovered. All such LTE-like sequences and DNA insulator sequences are encompassed by the present invention. Promoter

[0119] Within an expression cassette inserted into the viral vector as described herein, there is at least a promoter sequence, wherein the expression of the transgene can be controlled by the promoter.

[0120] The promoter may comprise DNA sequence starting at least 2 kb, preferably 3 kb, more preferably 4 kb upstream to the initiation site of the transgene. These sequences preferably contain known promoter sequence elements, such as specific transcription binding sites, and distal sequences upstream of the gene, containing additional regulatory elements. The promoter useful in the invention can be any promoter desired to control / regulate the expression of a transgene. Also, other known tissue-specific or cell-specific promoters may be used, such as neuron-specific promoters.

[0121] In some embodiments, the promoter contemplated is a constitutive mammalian promoter as are known in the art (e.g., SV40, CMV, CAG, EFla, UbC, RSV, 0-actin, PGK, and the like), but other promoters (as discussed herein and otherwise known in the art) can be used, if desired.

[0122] In some embodiments, the promoter useful in the invention can be an ubiquitous promoter as are known in the art. By “ubiquitous promoters” or “non-specific promoters” it is meant herein that the promoters are active in a wide range of cells, tissues, and / or organs.

[0123] In some embodiments, the promoter contemplated is an inducible promoter as are known in the art.

[0124] The viral expression vector of the invention is directed more particularly to vertebrate, preferably to mammals, more preferably primates and humans. Therefore, those skilled in the art will recognize that such promoters are specific to species and would be able to select homologous sequences of a particular species of interest.

[0125] Pharmaceutical Composition

[0126] The viral vector as described in this 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 typically will be liquid, but also can be solid, or a combination of liquid and solid components. The carrier desirably is a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). The composition can further comprise any other suitable components, especially for enhancing the stability of the composition and / or its end-use. Accordingly, there is a wide variety of suitable formulations of the composition of the viral vector. The following formulations and methods are merely exemplary and are in no way limiting.

[0127] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multidose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0128] In addition, the composition can comprise additional therapeutic or biologically-active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present. Factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the viral vector and physiological distress. Immune system suppressors can be administered with the composition to reduce any immune response to the vector itself or associated with a disorder. Alternatively, immune enhancers can be included in the composition to upregulate the body's natural defenses against disease. Antibiotics, i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders.

[0129] Administration

[0130] As used herein, the term “administration” or “administering” is defined to include an act of providing the viral vector as described herein or a pharmaceutical composition comprising the viral vector as described herein to a subject in performing the methods of the invention. Exemplary routes of administration include, but are not limited to, intrathecal administration, intra cerebro-ventricular, lumbar puncture, or striatum injection, as well as a combination thereof. In embodiments, the replication defective HSV vector is administered through one or more injections into the striatum. In embodiments, the viral vector as described herein is stereotaxi cally injected into the striatum. In embodiments, the replication defective HSV vector is administered through intrathecal administration.

[0131] The term “therapeutically effective amount” or “effective amount” means the amount of the viral vectors that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Thus, the term “therapeutically effective amount” is used herein to denote any amount of a formulation that causes a substantial improvement in a condition when applied to the affected areas repeatedly over a period of time. The amount will vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of formulation applied.

[0132] Determining a therapeutically or prophylactically effective amount of the delivery vector can be done based on animal data using routine computational methods. Appropriate doses will depend, among other factors, on the specifics of the transfer vector chosen, on the route of administration, on the number of injection sites, on the mammal being treated (e.g., human or non-human primate or other mammal), age, weight, and general condition of the subject to be treated, the severity of the disorder being treated, the location of the area within the heart being treated and the mode of administration. Thus, the appropriate dosage may vary from patient to patient.

[0133] Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. The dosage may need to be adjusted to take into consideration an alternative route of administration, decrease in expression efficacy over time, or to balance the therapeutic benefit against any side effects.

[0134] HSV-1 vectors can efficiently infect cells and resist immune clearance, which might be attributed to the innate immune-evasive properties of HSV tegument proteins. Natural immune- evading functions coupled with deletion of IE genes from the HSV-1 vector backbone (such as ICP4, ICP22, and ICP27) allow multiple doses of the defective viral vector derived from HSV-1 vectors to improve transduction efficiency and make the vector especially well-suited for gene therapy. (See Heldwein et al., Cell. Mol. Life Sci., 2008, 65, 1653-1668. Tognarelli et al., Front. Cell. Infect. Microbiol., 2019, 9, 127. Yang et al., Front. Immunol., 2019, 10, 2196. Gurevich et al., Nature Medicine, 2022, 28,780-788.) Methods

[0135] This application proposes a mechanism of action for using the viral vector as described herein (preferably, a replication defective viral vector derived from HSV-1) for the efficient delivery and expression of a transgene of interest in the brain of a subject in need thereof. Injection of the viral vector as described herein intrathecally or into the striatum will allow the vector to infect the nerve fibers in the brain, leading to the stable expression of the transgene in neurons driven by the promoters as described herein.

[0136] Preferably, the replication-defective HSV-1 of the invention provides for the delivery and expression of the transgene to one or more target cells of the brain. Preferably, the one or more target cells of the brain are selected from substantia nigra, striatum, and cortex, or combinations thereof. Preferably, when expressing the transgene in the cortex, the transgene is expressed in the prefrontal cortex.

[0137] Provided herein are also methods of delivering a transgene to the brain of a subject comprising administrating one or more doses of the replication-defected HSV vectors as described herein, or a pharmaceutical composition comprising thereof, to the subject. Preferably, the method comprises administering to the subject a pharmaceutical composition comprising a replication-defective herpes simplex type 1 virus (HSV-1) as described herein; wherein the pharmaceutical composition is administered intrathecally or by one or more injections into the striatum of the subject.

[0138] The method of delivering the transgene to the brain of a subject can further comprise the administration (i.e., pre-administration, co-administration, and / or post-administration) of other treatments and / or agents to modify (e.g., enhance) the effectiveness of the method. The method of the invention can further comprise the administration of other substances which locally or systemically alter (i.e., diminish or enhance) the effect of the composition on a host. For example, substances that diminish any systemic effect of the protein produced through expression of the nucleic acid sequence of the vector in a host can be used to control the level of systemic toxicity in the host. Likewise, substances that enhance the local effect of the protein produced through expression of the nucleic acid sequence of the vector in a host can be used to reduce the level of the protein required to produce a prophylactic or therapeutic effect in the host. Such substances include antagonists, for example, soluble receptors or antibodies directed against the protein produced through expression of the nucleic acid sequence of the vector, and agonists of the protein.

[0139] Also provided herein is a pharmaceutical composition useful for delivering a transgene to the brain of a subject. Preferably, the pharmaceutical composition comprises a replicationdefective herpes simplex type 1 virus (HSV-1) as described herein; wherein the pharmaceutical composition is suitable for intrathecal administration or for one or more injections into the striatum of the subject.

[0140] The pharmaceutical composition useful for delivering the transgene to the brain of a subject can be co-administered with (i.e., pre-administration, co-administration, and / or postadministration) other treatments and / or agents to modify (e.g., enhance) the effectiveness of the pharmaceutical composition. The other substances can locally or systemically alter (i.e., diminish or enhance) the effect of the composition on a host. For example, substances that diminish any systemic effect of the protein produced through expression of the nucleic acid sequence of the vector in a host can be used to control the level of systemic toxicity in the host. Likewise, substances that enhance the local effect of the protein produced through expression of the nucleic acid sequence of the vector in a host can be used to reduce the level of the protein required to produce a prophylactic or therapeutic effect in the host. Such substances include antagonists, for example, soluble receptors or antibodies directed against the protein produced through expression of the nucleic acid sequence of the vector, and agonists of the protein.

[0141] This application proposes a mechanism of action for using the viral vector as described herein (preferably, a replication defective viral vector derived from HSV-1) for the efficient delivery and expression of a transgene of interest in the brain of a subject in need thereof. Injection of the viral vector as described herein intrathecally or into the striatum will allow the vector to infect the nerve fibers in the brain, leading to the stable expression of the transgene in neurons driven by the promoters as described herein.

[0142] The following examples are intended to illustrate but not limit the invention. All citations throughout the disclosure are hereby expressly incorporated by reference.

[0143] Examples

[0144] There are reports of multiple immediate- early gene-deficient herpes simplex virus vectors allowing efficient gene delivery to neurons in culture and widespread gene delivery to the central nervous system in vivo. Lilley CE, Groutsi F, Han Z, Palmer JA, Anderson PN, Latchman DS, Coffin RS. J Virol. 2001 May;75(9):4343-56. doi: 10.1128 / JVI.75.9.4343-4356.2001.

[0145] A defective viral vector according to the invention, which lacks a combination of essential and non-essential genes and includes a reporter gene (mGreen Lantern) under the control of EFla promoter and inserted in the LAT region (“HSV-1A”), was stereotaxi cally injected in the striatum of Balb / c mice (4.4E+06 PFU in 2 uL). The mice were anesthetized by isoflurane (4%, for induction), in an induction chamber coupled with a vaporizer and to an oxygen concentrator and were placed on the stereotaxic frame. Anesthesia was maintained by isoflurane (2%) with a face mask coupled to the isoflurane vaporizer and oxygen concentrator machine. The skull was exposed and a hole was drilled. A total of 2 pl of viral solution (stock) was injected, unilateral only, at the following coordinates: A-P,-0.4 mm; M-L, +2.5 mm; D-V, -3.2 mm. Depth of anesthesia and rectal temperature was verified every 5 minutes. After the injection, the skin was sutured and the mice were allowed to recover before being placed back in the cage.

[0146] The mice were sacrificed after 3 days or 10 days post injection. The brains were collected and sectioned for immunofluorescence imaging.

[0147] Expression of mGreen lantern was visible at 3 days and 10 days at the striatum (injection site) and also in the substantia nigra, subthalamic nucleus, ependymal cells of the lateral ventricle and cortex, which demonstrated axonal transport (e.g., that the HSV-1A vector transduced neurons at site of administration but also projecting neurons following retrograde transport through the axon) in CNS. (see Fig. 1A through Fig. 1C and Fig. 3) mGreen lantern expression was colocalized with the neuronal marker NeuN which showed that mGreen lantern is mainly expressed in neurons in striatum, (see Fig. 2)

[0148] Additionally, the replication defective viral vector of the invention efficiently targeted projecting neurons of the substantia nigra and cortex following administration into the striatum 3 days after injection, showing, for the first time, transgene expression in pre-frontal cortex from striatum injection (see Fig. 3).

[0149] Sagittal brain sections were immunostained for mGreen lantern, for the dopaminergic neuron marker Tyrosine hydroxylase (TH) and the nuclear marker DAPI. mGreen lantern was colocalized with TH staining, which showed that the HSV1 vector transduced dopaminergic neurons of the substantia nigra after intra-striatal administration, (see Fig. 4) An HS V 1 -derived vector with mGreen lantern under the control of CAG promoter (“HSV-1B”) was administered in the striatum of the right hemisphere of the mouse brain (4.4E+06 PFU in 2 uL). After 3 days, brains were collected and sectioned for immunofluorescence imaging. mGreen lantern direct fluorescence was imaged. mGreen lantern was also immunostained with an anti-GFP antibody (which recognizes the mGreen lantern protein). Cell nuclei were stained with Hoechst. mGreen lantern expression was confirmed in cortical neurons in injected hemisphere and was also observed in neurons in the cortex of the contralateral non-injected hemisphere. This demonstrated that the HSV-1B vector also transduced cortical neurons projecting in striatum from the contralateral hemisphere, (see Fig. 5) Additionally, kinetics of mGreen lantern expression in cortical neurons was investigated at 1 and 6 weeks following intra-striatal administration in mice of HSV-1B (4,4E+06 PFU). The number of cortical neurons expressing mGreen lantern was similar between 1 and 6 weeks, which demonstrated stable mGreen lantern expression following retrograde transport of the vector, (see Fig. 6A). Fig. 6B shows the quantification of the number of vector episomes in the cortex as determined by ddPCR. The number of episomes was stable between 1 and 6 weeks. Fig. 6C depicts the quantification of number of mGreen lantern transcripts (RNA) in the cortex. mGreen lantern expression was stable between 1 and 6 weeks as determined by reverse transcription-ddPCR. Mean + / - SD. Each dot represents one injected hemisphere.

[0150] Both the HSV-1 A and HSV-1B vectors were administered in the striatum of mouse brain (4,4E+06 PFU in 2 uL). After 3 days, brains were collected and sectioned for immunofluorescence imaging. mGreen lantern direct fluorescence was imaged and mGreen lantern was also immunostained with an anti-GFP antibody which recognizes the mGreen lantern protein. Cell nuclei were stained with Hoechst. mGreen lantern expression was seen at similar levels in the cortex after intra-striatal injection of both vectors demonstrating that both EFla and CAG promoters drove mGreen lantern expression in cortical neurons. (See Fig. 7)

[0151] Table 1 compares the results obtained for HSV-1 A against published data obtained from the injection into the striatum with other known defective viral vectors (JANI6 and 1764A27A4) A non-defective viral vector (1716-LAT-hGUSB) was provided as a control. Table 1 Gene expression from HSV-1 vectors following infection in brain.

[0152] NR = non- replicative; (3) VP 16 is present but contains a disabling mutation in the transactivating domain. (4) Somatosensory cortex, visual cortex, striatum, dorsal hippocampus, cisterna magna and lateral ventricle. (5) Regions of transgene activity varied by injection site and included cerebral cortex, striatum, thalamus, hypothalamus, substantia nigra, hippocampus, midbrain, pons, medulla, cerebellum, spinal cord.

Claims

CLAIMSWhat is claimed:

1. A method of delivering a transgene to the brain of a subject, the method comprising administering to the subject a pharmaceutical composition comprising a replication-defective herpes simplex type 1 virus (rdHSV-1) vector and a pharmaceutically acceptable carrier, wherein the rdHSV-1 vector comprises:(i) an inactivating mutation of one copy of infected cell protein 0 (ICPO) HSV gene; and(ii) the transgene; wherein the rdHSV-1 vector expresses a functional ICPO protein; and wherein the pharmaceutical composition is administered intrathecally or by one or more injections into the striatum of the subject.

2. The method according to claim 1 , wherein the inactivating mutation of the one copy of the IPCO gene is an inactivating mutation in the copy of ICPO in the “LAT, ICPO, UL34.5 cluster” from the IRL region of the HSV.

3. The method according to claim 1 or 2, wherein the transgene is part of a transcription cassette, preferably wherein the transcription cassette is introduced into the LAT locus.

4. The method according to any one of claims 1-3, wherein the replication-defective HSV-1 vector further comprises an inactivating mutation in infected cell protein 4 (ICP4) and infected cell protein 27 (ICP27).

5. The method according to claim 4, wherein the replication-defective HSV-1 vector comprises an inactivating mutation in both copies of ICP4.

6. The method according to any one of claims 1-5, wherein the replication-defective HSV-1 vector further comprises an inactivating mutation in one or more genes encoding non-essential proteins selected from an infected cell protein 22 (ICP22), infected cell protein 34.5 (ICP34.5), long unique region (UL) UL41, UL55, and UL56, or combinations thereof.

7. The method according to claim 6, wherein the replication-defective HSV-1 vector further comprises an inactivating mutation in ICP22, ICP34.5, UL41, UL55, and UL56.

8. The method according to any one of claims 1-7, wherein the replication-defective HSV-1 vector has reduced cytotoxicity as compared to a wild-type herpes simplex type 1 virus.

9. The method according to any one of claims 1-8, wherein the replication-defective HSV-1 vector is suitable for delivering the transgene to one or more target cells of the brain.

10. The method according to claim 9, wherein the one or more target cells of the brain are selected from substantia nigra, striatum, and cortex, or combinations thereof.

11. The method according to claim 9, wherein the replication-defective HSV-1 vector is suitable for delivering the transgene to the prefrontal cortex.

12. The method according to any one of claims 1-11, wherein the composition is administered intrathecally.

13. The method according to any one of claims 1-11, wherein the composition is administered through one or more injections into the striatum.

14. The method according to claim 13, wherein the composition is injected stereotaxically.

15. The method according to any one of claims 1-14, wherein the subject is a human.

16. A pharmaceutical composition useful for delivering a transgene to the brain of a subject, the pharmaceutical composition comprising a replication-defective herpes simplex type 1 virus (rdHSV-1) vector and a pharmaceutically acceptable carrier, wherein the (rdHSV-1) vector comprises:(i) an inactivating mutation of one copy of infected cell protein 0 (ICPO) HSV gene; and(ii) the transgene; wherein the rdHSV-1 vector expresses a functional ICPO protein; and wherein the pharmaceutical composition is suitable for intrathecal administration or for one or more injections into the striatum of the subject.

17. The composition for use according to claim 16, wherein the inactivating mutation of the one copy of the IPCO gene is an inactivating mutation in the copy of ICPO in the “LAT, ICPO, UL34.5 cluster” from the IRL region of the HSV.

18. The composition for use according to claim 16 or 17, wherein the transgene is part of a transcription cassette, preferably wherein the transcription cassette is introduced into the LAT locus.

19. The composition for use according to any one of claims 16-18, wherein the replicationdefective HSV-1 vector further comprises an inactivating mutation in infected cell protein 4 (ICP4) and infected cell protein 27 (ICP27).

20. The composition for use according to claim 19, wherein the replication-defective HSV-1 vector comprises an inactivating mutation in both copies of ICP4.

21. The composition for use according to any one of claims 16-20, wherein the replicationdefective HSV-1 vector further comprises an inactivating mutation in one or more genes encoding non-essential proteins selected from in infected cell protein 22 (ICP22), infected cell protein 34.5 (ICP34.5), long unique region (UL) UL41, UL55, and UL56, or combinations thereof.

22. The composition for use according to claim 21, wherein the replication-defective HSV-1 vector further comprises an inactivating mutation in ICP22, ICP34.5, UL41, UL55, and UL56.

23. The composition for use according to any one of claims 16-22, wherein the replicationdefective HSV-1 vector has reduced cytotoxicity as compared to a wild-type herpes simplex type 1 virus.

24. The composition for use according to any one of claims 16-23, wherein the composition is suitable for delivering the transgene to one or more target cells of the brain.

25. The composition for use according to claim 24, wherein the one or more target cells of the brain are selected from substantia nigra, striatum, and cortex, or combinations thereof.

26. The composition for use according to claim 24, wherein the composition is suitable for delivering the transgene to the prefrontal cortex.

27. The composition for use according to any one of claims 16-26, wherein the composition is administered intrathecally.

28. The composition for use according to any one of claims 16-26, wherein the composition is administered through one or more injections into the striatum.

29. The composition for use according to claim 28, wherein the composition is injected stereotaxi cally.

30. The composition for use according to any one of claims 16-29, wherein the subject is a human.