EP4007609A1 - Thérapie génique ciblée pour traiter des maladies neurologiques - Google Patents

Thérapie génique ciblée pour traiter des maladies neurologiques

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Publication number
EP4007609A1
EP4007609A1 EP20758033.3A EP20758033A EP4007609A1 EP 4007609 A1 EP4007609 A1 EP 4007609A1 EP 20758033 A EP20758033 A EP 20758033A EP 4007609 A1 EP4007609 A1 EP 4007609A1
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European Patent Office
Prior art keywords
vector
gene
mammal
targeted
viral vector
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EP20758033.3A
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German (de)
English (en)
Inventor
Michael G. Kaplitt
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Cornell University
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Cornell University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • Gene therapy has shown great promise in human patients for treating neurological diseases.
  • One advantage of invasive injection of gene therapy agents is the focal targeting of relevant train regions to prevent off target effects that often limit efficacy and cause side effects from drugs delivered throughout the brain. It is increasingly recognized, however, that targeting specific circuits can not only provide a greater level of specificity compared with targeting brain regions, but that resulting behaviors can differ when a specific circuit underlying that behavior is manipulated in isolation.
  • One way to limit expression to specific subpopulations of neurons is to use cell-type specific promoters, but these are often unreliable and still do not provide anatomic circuit specificity but rather restrict expression to all neurons expressing the gene driven by the chosen promoter. Therefore, anatomic targeting methods are desirable to manipulate populations of neurons which project from one brain region to regulate neurons in another brain region, referred to here as circuits.
  • a gene for a light-sensitive ion channel is delivered to a brain region, usually by a viral vector, and then the ion channel is expressed in all transduced neurons within a brain region capable of supporting expression from the given promoter.
  • a light probe is then placed within a target region so that the only neurons which will be activated are those which received the gene into the cell bodies in the first brain target and light to the axons in the second brain target.
  • this requires an implanted light probe and cannot be regulated non-invasively, and the safety of chronic light in the brain remains unknown.
  • a method for targeting a specific population of neurons in a mammal comprises delivery (administration) of a first viral vector with a conditionally activatable (non- functional until activated) gene or gene product to a mammalian structure to be modulated, e.g., which contains or is in proximity to axons from regulatory neurons, and delivery of a second viral vector expressing a gene, the product of which can activate the gene in the first viral vector, e.g., the second viral vector may be delivered to a different region containing a subset of the cell bodies that send the axons to the structure to be modulated.
  • the method comprises delivery of a viral vector with a conditionally activatable gene or gene product to cell bodies that send the axons to a structure to be modulated and delivery of a second vector expressing a gene, the product of which can activate the gene in the first viral vector, e.g., to the structure to be modulated.
  • the method comprises delivery of a first viral vector with a conditionally activatable gene or gene product, which first vector is capable of retrograde uptake into neurons, and delivery of a second vector expressing a gene, the product of which can activate the first viral vector.
  • the method comprises delivery of a first viral vector with a conditionally activatable gene or gene product, and delivery of a second vector expressing a gene, the product of which can activate the first viral vector, which second vector is capable of retrograde uptake into neurons. , to a different region containing a subset of the cell bodies that send the axons to the structure to be modulated.
  • the structure to be modulated is a peripheral organ or a brain region.
  • the viral vector is an adeno- associated virus, adenovirus, canine adenovirus, herpes simplex virus, or lentivirus vector, e.g., a retrograde form of adeno-associated virus, lentivirus or canine adenovirus.
  • Molecules that provide for retrograde forms of vectors are known to the art and include but are not limited to native viral proteins, such as HSV protein, rabies virus G, glycoprotein type C, VSV G, B19G, pseudorabies virus protein, AAV capsid protein, and dynein.
  • the first vector contains recognition sites capable of recombination and activation of the gene, e.g., a therapeutic gene, in response to a recombinase delivered by the second vector.
  • the recombinase is Cre or Flp.
  • a recombinase enzyme is conditionally activated by an exogenous chemical or stimulus.
  • the first vector contains a gene that does not express a functional protein autonomously
  • the second vector expresses a protein that is capable of activating gene expression or protein function from the first gene.
  • the second vector expresses regulatable or autonomously active transcription factors capable of transactivating expression of the gene, e.g., encoding a therapeutic gene product, from the first vector.
  • the viral vector is delivered via direct infusion into the organ or brain region. In one embodiment, the viral vector is delivered through focal disruption of the blood-brain barrier using focused ultrasound. In one embodiment, the viral vector is delivered to one brain region in a single treatment, followed by delivery of a second viral vector to a second brain region in a second treatment. In one embodiment, the two treatments are separated by at least 24, 36 or 72 hours or more. In one embodiment, the two treatments are sequential, e.g., occur on the same day. In one embodiment, the two treatments are conducted simultaneously. In one embodiment, the two treatments are separated by 1, 2 or 3 weeks or more.
  • a system or a kit comprising a first vector with a non- functional (conditionally activatable) gene or gene product, and a second vector expressing a gene, the product of which can activate tire gene in the first vector, wherein optionally one of the vectors is capable of retrograde uptake in neurons.
  • the vectors are both viral vectors, e.g., AAV vectors that may differ in serotype.
  • the first vector encodes an ion channel protein.
  • the first vector encodes a G coupled protein receptor ion channel protein.
  • the first vector encodes a transcription factor.
  • one vector comprises an open reading frame for a selected (first) gene product and the other vector comprises an open reading frame for a selected (second) gene product. Expression of one of the gene products is conditionally activatable by the other gene product. For example, one of the open reading frames is flanked by recombination sites for a recombinase that is encoded by the other vector.
  • one of the vectors is a recombinant viral vector, e.g., rAAV vector.
  • both of the vectors are recombinant viral vectors.
  • when both of the vectors are recombinant viral vectors one is a retrograde viral vector.
  • non-invasive delivery of two vectors is employed.
  • the delivery of the vectors is sequential.
  • one (first) vector is systemically delivered, e.g., via injection, and focused ultrasound is used to target that vector to a specific site in the central nervous system.
  • the other (second) vector is systemically delivered, e.g., via injection, and focused ultrasound is used to target that vector to a specific (e.g., different) site in the central nervous system.
  • focused ultrasound is used to target that vector to the same site as the first vector.
  • invasive delivery of two vectors is employed.
  • the delivery of the vectors is on the same day.
  • the delivery of the vectors is sequential, e.g., separated by hours, days or weeks.
  • the vectors are delivered to the same site.
  • the vectors are delivered to different sites.
  • non-invasive delivery of one vector and invasive delivery of another vector are employed.
  • the delivery of the vectors is on the same day.
  • the delivery of the vectors is sequential.
  • the vectors are delivered to the same site.
  • the vectors are delivered to different sites.
  • FIG. 1 Sagittal section through rat brain. Viral vectors are delivered as depicted through either MRgFUS or stereotactic direct injection.
  • FIG. 1 Stereotactic injection of AAV into rat brain. Immunostaining for mCherry (reporter gene), GFP (reporter gene), TH (tyrosine hydroxylase - dopaminergic neurons marker) and D API (nuclear marker) following unilateral and ipsilateral administration of AAVl/2.DIO.hM3Dq.mCherry into striatum and RetroAAVl/2.Cre.GFP into substantia nigra.
  • Figure 3 Stereotactic injection of AAV into rat brain.
  • mCherry reporter gene
  • GFP reporter gene
  • TH tyrosine hydroxylase - dopaminergic neurons marker
  • D API nuclear marker
  • mCherry reporter gene
  • GFP reporter gene
  • TH tyrosine hydroxylase-dopaminergic neurons marker
  • DAPI nuclear marker
  • mCherry reporter gene
  • GFP reporter gene
  • TH tyrosine hydroxylase - dopaminergic neurons marker
  • DAPI nuclear marker
  • A“vector” refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide, and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo.
  • Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles.
  • the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest), a coding sequence of interest in vaccine development (such as a polynucleotide expressing a protein, polypeptide or peptide suitable for eliciting an immune response in a mammal), and/or a selectable or detectable marker.
  • Transduction,”“transfection,”“transformation” or“transducing” as used herein are terms referring to a process for the introduction of an exogenous polynucleotide into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant virus to introduce the exogenous polynucleotide to the host cell.
  • Transduction, transfection or transformation of a polynucleotide in a cell may be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), e.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays.
  • Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation and electroporation, as well as other non-viral gene delivery techniques.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Gene delivery refers to the introduction of an exogenous
  • polynucleotide into a cell for gene transfer may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.
  • Gene transfer refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.
  • Gene expression or“expression” refers to the process of gene transcription, translation, and post-translational modification.
  • An“infectious” virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic.
  • the term does not necessarily imply any replication capacity of the virus.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • An“isolated” polynucleotide e.g., plasmid, virus, polypeptide or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Isolated nucleic acid, peptide or polypeptide is present in a form or setting that is different from that in which it is found in nature.
  • a given DNA sequence e.g., a gene
  • RNA sequences such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins.
  • the isolated nucleic acid molecule may be present in single-stranded or double- stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the molecule will contain at a minimum the sense or coding strand (i.e., the molecule may single-stranded), but may contain both the sense and anti-sense strands (i.e., the molecule may be double-stranded).
  • Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments may be preferred. Thus, for example, a 2-fold enrichment, 10-fold enrichment, 100- fold enrichment, or a 1000-fold enrichment.
  • A“transcriptional regulatory sequence” refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked.
  • Transcriptional regulatory sequences of use in the present disclosure generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.
  • “Operably linked” refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner.
  • a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence.
  • An operably linked TRS is generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.
  • Heterologous means derived from a genotypically distinct entity from the entity to which it is compared.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • a transcriptional regulatory element such as a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element.
  • A“terminator” refers to a polynucleotide sequence that tends to diminish or prevent read-through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator).
  • the degree to which transcription is disrupted is typically a function of the base sequence and/or the length of the terminator sequence.
  • particular DNA sequences generally referred to as“transcriptional termination sequences” are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and/or disengage from the DNA being transcribed.
  • sequence-specific terminators include polyadenylation
  • poly A sequences e.g., SV40 polyA.
  • insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated.
  • Terminators may thus prevent transcription from only one direction (“uni-directional” terminators) or from both directions (“bi-directional” terminators), and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both.
  • sequence-specific termination sequences or sequence-non-specific terminators or both.
  • “Host cells,”“cell lines,”“cell cultures,”“packaging cell line” and other such terms denote higher eukaryotic cells, such as mammalian cells including human cells, useful in the present disclosure, e.g., to produce recombinant virus or recombinant fusion polypeptide. These cells include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • A“control element” or“control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in tire art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. Promoters include AAV promoters, e.g., P5, PI 9, P40 and AAV ITR promoters, as well as heterologous promoters.
  • An“expression vector” is a vector comprising a region which encodes a gene product of interest, and is used for effecting the expression of the gene product in an intended target cell.
  • An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target.
  • the combination of control dements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
  • polypeptide and“protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component.
  • exogenous when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means.
  • An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell.
  • an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature, e.g., an expression cassette which links a promoter from one gene to an open reading frame for a gene product from a different gene.
  • Transformed or transgenic is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence.
  • the host cells are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector.
  • sequence homology means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less, or 2 bases or less.
  • the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); not less than 9 matches out of 10 possible base pair matches (90%), or not less than 19 matches out of 20 possible base pair matches (95%).
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less or 2 or less.
  • two protein sequences are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater.
  • the two sequences or parts thereof are more homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
  • the term“corresponds to” is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 85%, 90%, 95% or more, e.g., 99% or 100%, sequence identity.
  • the term“complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence“TATAC’ corresponds to a reference sequence‘TATAC” and is complementary to a reference sequence “GTATA”.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • the term“percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I
  • substantially identical denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • Constant amino acid substitutions are, for example, aspartic- glutamic as polar acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucineZisoleudne/methionine/valine/alanine/glycine/proline as non-polar or hydrophobic amino acids; serine/ threonine as polar or uncharged hydrophilic amino acids.
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side drains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side drains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic; tip, tyr, phe.
  • the disclosure also envisions polypeptides with non-conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another.
  • the disclosure provides a gene transfer vector, e.g., a viral gene transfer vector, useful to deliver genes to neurons or nerve fibers.
  • a gene transfer vector e.g., a viral gene transfer vector
  • Various aspects of the gene transfer vector and method are discussed below. Accordingly, any combination of parameters can be used according to the gene transfer vector and the method.
  • A“gene transfer vector” is any molecule or composition that has the ability to carry a heterologous nucleic arid sequence into a suitable host cell where synthesis of the encoded protein takes place.
  • a gene transfer vector is a nucleic acid molecule that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate the heterologous nucleic acid sequence.
  • the gene transfer vector is comprised of DNA.
  • suitable DNA-based gene transfer vectors include plasmids and viral vectors.
  • gene transfer vectors that are not based on nucleic acids, such as liposomes are also known and used in the art.
  • the gene transfer vector can be based on a single type of nucleic acid (e.g., a plasmid) or non-nucleic acid molecule (e.g., a lipid or a polymer).
  • the gene transfer vector can be integrated into the host cell genome, or can be present in the host cell in the form of an episome.
  • the gene transfer vector is a viral vector.
  • Suitable viral vectors include, for example, retroviral vectors, herpes simplex virus (HSV)-based vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors, AAV -adenoviral chimeric vectors, and adenovirus-based vectors.
  • HSV herpes simplex virus
  • AAV adeno-associated virus
  • AAV -adenoviral chimeric vectors e.g., AAV -adenoviral chimeric vectors
  • adenovirus-based vectors e.g., adeno-associated virus (AAV)-based vectors.
  • AAV adeno-associated virus
  • the disclosure provides an adeno-associated virus (AAV) vector.
  • the AAV vector may include a gene to be expressed and additional components that do not materially affect the AAV vector (e.g., genetic elements such as poly(A) sequences or restriction enzyme sites that facilitate manipulation of the vector in vitro).
  • Adeno-associated virus is a member of the Parvoviridae family and comprises a linear, single-stranded DNA genome of less than about 5,000 nucleotides.
  • AAV requires co-infection with a helper virus (i.e., an adenovirus CM- a herpes virus), or expression of helper genes, for efficient replication.
  • AAV vectors used for administration of therapeutic nucleic acids typically have approximately 96% of the parental genome deleted, such that only the terminal repeats (ITRs), which contain recognition signals for DNA replication and packaging, remain. This eliminates immunologic or toxic side effects due to expression of viral genes.
  • ITRs terminal repeats
  • delivering specific AAV proteins to producing cells enables integration of the AAV vector comprising AAV ITRs into a spedfic region of the cellular genome, if desired (see, e.g.,
  • Host cells comprising an integrated AAV genome show no change in cell growth or morphology (see, for example, U.S. Patent 4,797,368).
  • the AAV ITRs flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)).
  • the terminal 145 nucleotides are self- complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication by serving as primers for the cellular DNA polymerase complex.
  • the Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the pl9 promoter.
  • the Rep78 and Rep68 proteins are multifunctional DNA binding proteins that perform helicase and nickase functions during productive replication to allow for the resolution of AAV termini (see, e.g., Im et al., Cell. 61:447 (1990)). These proteins also regulate transcription from endogenous AAV promoters and promoters within helper viruses (see, e.g., Pereira et al., J. Virol.. 71: 1079 (1997)). The other Rep proteins modify the function of Rep78 and Rep68.
  • the cap genes encode the capsid proteins VP1 , VP2, and VP3. The cap genes are transcribed from the p40 promoter.
  • the AAV vector may be generated using any AAV serotype known in the art.
  • AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or nonhuman primate tissues (reviewed in, e.g., Wu et al., Molecular Therapy. 14(3): 316 (2006)).
  • the AAV serotypes have genomic sequences of significant homology at the nucleic acid sequence and amino acid sequence levels, such that different serotypes have an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms.
  • AAV serotypes 1-6 and 7-9 are defined as“true” serotypes, in that they do not efficiently cross-react with neutralizing sera specific for all other existing and characterized serotypes.
  • AAV serotypes 6, 10 (also referred to as RhlO), and 11 are considered“variant” serotypes as they do not adhere to the definition of a“true” serotype.
  • AAV serotype 2 (AAV2) has been used extensively for gene therapy applications due to its lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression (see, e.g., Carter, Hum. Gene Ther.. 16:541 (2005); and Wu et al., supra).
  • Genome sequences of various AAV serotypes and comparisons thereof are disclosed in, for example, GenBank Accession numbers U89790, J01901 , AF043303, and AF085716; Chiorini et al., J. Virol.. 71:6823 (1997); Srivastava et al., J. Virol.. 45:555 (1983); Chiorini et al., J. Virol..
  • AAV rep and ITR sequences are particularly conserved across most AAV serotypes.
  • the Rep78 proteins of AAV2, AAV3A, AAV3B, AAV4, and AAV6 are reportedly about 89-93% identical (see Bantel-Schaal et al., J. Virol. 73(2):939 (1999)). It has been reported that AAV serotypes 2, 3A, 3B, and 6 share about 82% total nucleotide sequence identity at the genome level
  • rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (e.g., functionally substitute) corresponding sequences from other serotypes during production of AAV particles in mammalian cells.
  • the cap proteins which determine the cellular tropism of the AAV particle, and related cap protein-encoding sequences, are significantly less conserved than Rep genes across different AAV serotypes.
  • the AAV vector can comprise a mixture of serotypes and thereby be a “chimeric” or“pseudotyped” AAV vector.
  • a chimeric AAV vector typically comprises AAV capsid proteins derived from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes.
  • a pseudotyped AAV vector comprises one or more ITRs of one AAV serotype packaged into a capsid of another AAV serotype.
  • Chimeric and pseudotyped AAV vectors are further described in, for example, U.S. Patent No. 6,723,551; Flotte, Mol. Ther.. 13(1):1 (2006); Gao et al., J. Virol. 78:6381 (2004); Gao et al., Proc. Natl. Acad. Sci. USA. 99:11854 (2002); De et al., Mol. Ther.. 13:67 (2006); and Gao et al., Mol. Ther.. 13:77 (2006).
  • the AAV vector is generated using an AAV that infects humans (e.g., AAV2).
  • the AAV vector is generated using an AAV that infects non-human primates, such as, for example, the great apes (e.g., chimpanzees), Old World monkeys (e.g., macaques), and New World monkeys (e.g., marmosets).
  • the AAV vector is generated using an AAV that infects a non-human primate pseudotyped with an AAV that infects humans. Examples of such pseudotyped AAV vectors are disclosed in, e.g., Cearley et al., Molecular Therapy. 13:528 (2006).
  • an AAV vector can be generated which comprises a capsid protein from an AAV that infects rhesus macaques pseudotyped with AAV2 inverted terminal repeats (ITRs).
  • the inventive AAV vector comprises a capsid protein from AAV10 (also referred to as“AAVrh.10”), which infects rhesus macaques pseudotyped with AAV2 ITRs (see, e.g., Watanabe et al., Gene Ther.. 17(8):1042 (2010); and Mao et al., Hum. Gene Therapy. 22:1525 (2011)).
  • the AAV vector may comprise expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the nucleic acid sequence in a host cell.
  • expression control sequences such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the nucleic acid sequence in a host cell.
  • Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA. (1990).
  • promoters including constitutive, inducible, and repressble promoters, from a variety of different sources are well known in the art.
  • Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources.
  • Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3 * or 5’ direction).
  • Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter.
  • Inducible promoters include, for example, the Tet system (U.S. Patent Nos. 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci..
  • Enhancers refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many ldlobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences. In one embodiment, the nucleic acid sequence is operably linked to a CMV
  • enhancer/chicken beta-actin promoter also referred to as a“CAG promoter”
  • CAG promoter see, e.g., Niwa et al., Gene. 108:193 (1991); Daly et al., Proc. Natl Acad. Sci. U.S.A..26:2296 (1999); and Sondhi et al., Mol. Ther.. 15:481 (2007)).
  • AAV vectors are produced using well characterized plasmids.
  • human embryonic kidney 293T cells are transfected with one of the transgene specific plasmids and another plasmid containing the adenovirus helper and AAV rep and cap genes (specific to AAVrh.10, 8 or 9 as required). After 72 hours, the cells are harvested and the vector is released from the cells by five freeze/thaw cycles. Subsequent centrifugation and benzonase treatment removes cellular debris and unencapsidated DNA. Iodixanol gradients and ion exchange columns may be used to further purify each AAV vector. Next, the purified vector is concentrated by a size exclusion centrifuge spin column to the required concentration.
  • the buffer is exchanged to create the final vector products formulated (for example) in lx phosphate buffered saline.
  • the viral titers may be measured by TaqMan ® real-time PCR and the viral purity may be assessed by SDS-PAGE.
  • Striatum is formed by different populations of neurons, which receive and send information to multiple regions in the cerebral cortex and limbic system. Injecting a vector, e.g., an AAV-mediated gene therapy vector, directly into the striatum would result in tire expression of gene of interest in all neurons in the targeted area, regardless of their function and connections to other brain structures. This action would result a more nonspecific response in the targeted area, not always beneficial for the evolution of the disease.
  • the present disclosure provides for targeted delivery of a pair of vectors, e.g., targeted to striatal neuronal populations connected to the substantia nigra. For example, dopaminergic neurons in substantia nigra project to the striatum and a
  • RetroAAV.Cre virus delivered in the substantia nigra would migrate via axons and express Cre recombinase in the striatum in a specific neuronal population. Even though the AAV delivered to the striatum would infect all the cells in the area, only tire natrons that receive input from the substantia nigra would express the Cre recombinase required for the expression of the AAV-mediated gene of interest. This allows for delivery and manipulation of genes of interest in specific neuronal populations of neuronal circuits in the brain, optionally in a less invasive manner for delivery to the brain than disrupting the skull.
  • Any method that allows for targeted delivery of the vectors may be employed.
  • any method that allows for targeted delivery of one vector to one site and targeted delivery of the other vector to another site may be employed.
  • a pair of rAAV vectors is employed.
  • One of the pairs is a retro AAV that encodes a recombinase, e.g., Cre.
  • the retroAAV has an AAV2 capsid having at least one or two substitutions that allow for uptake by nerve terminals in a given brain region, after which it is then transported back to their cell bodies.
  • the retroAAV is taken up by the neurons of the striatum that project into the substantial nigra.
  • An exemplary retroAAV capsid sequence is:
  • the other (non-retro) rAAV of the pair comprises sequences for a gene product of interest that is not expressed due to placement of sites for the recombinase.
  • the second vector has an AAV 1/2 hybrid capsid that is not taken up retrograde.
  • the non-retro rAAV when introduced to the striatum, it only transduces neurons of the striatum and is not taken up into the neurons that project into the striatum from elsewhere.
  • At least one or both of the pair of rAAVs is non- invasively administered, e.g., via intravenous injection into the bloodstream, followed up opening of the blood-brain barrier (BBB) in the relevant target region by MRgFUS so that the virus can get from the bloodstream into that region to transduce those cells.
  • BBB blood-brain barrier
  • Figure 1 AAV 1/2 with an inactive floxed gene was delivered by intravenous injection into the striatum using MRgFUS targeted to the striatum to focally and transiently open the blood brain barrier. Under normal circumstances, an active gene would be expressed everywhere in the striatum.
  • a second vector is present in the same neurons and expresses the recombinase, e.g., cre-recombinase, to flip the construct to the active form to allow for gene expression.
  • a second treatment was given, this time with the retroAAV expressing either a recombinase, e.g., Cre, injected into the bloodstream IV and MRgFUS BBB disruption performed in the substantia nigra, so that the retroAAV would only enter the substantia nigra since the BBB was closed in the striatum.
  • MRgFUS treatments are separated by at least 24 hours to allow the BBB to close in the first area, permitting targeted delivery to the second area.
  • retroAAV would be taken up into the neurons from the striatum that project into the nigra.
  • retroAAV-cre is taken up into striatal neurons pojecting to the nigra from the striatum, those which already received the inactive floxed gene from the first stratal delivery would then become activated because they would now express Cre, allowing for gene activation.
  • Delivery of retroAAV to the nigra which does not express the appropriate recombinase, e.g., expresses GFP would fail to activate gene expression. See Figures 4 and 5, confirming that gene expression was targeted exclusively to stratal neurons which project to the substantia nigra.
  • invasive delivery may be used to deliver one or more of the vectors, e.g., rAAVs.
  • Invasive deliver includes using surgery to make a hole in the skull and insert a needle into these two targets to directly infuse the respective vectors. That can be done in a single session, rather than two sessions which are used for MRgFUS, since one virus is administered into one region, e.g., via injection, and the other virus into another region.
  • direct surgical injection delivers one of the viruses to one target site and MRgFUS is used to deliver the other virus to another target site.
  • one region (A) is targeted with the inactive vector and another region (B) is targeted with a retrograde vector expressing the activating gene product (such as Cre).
  • one region (A) has the activating gene in a non-retrograde vector and the other region (B) has the inactive gene in a retrograde vector.
  • Cre may be expressed from AAV1/2 delivered to the striatum, followed by delivery of the floxed-DREADD construct in retroAAV to the substantia nigra.
  • a recombinase encoding vector (a non-retro vector) may be delivered via MRgFUS to the striatum after the desired gene product encoding sequence, e.g., DREADD, is delivered to the striatum, e.g., via MRgFUS.
  • DREADD desired gene product encoding sequence
  • the pair of vectors may be employed to prevent, inhibit or treat a neurological disease or disorder via neurons.
  • dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbent (NAc) are targeted or VTA projections to medial prefrontal cortex (mPFC) are targeted.
  • VTA-NAc promotes depression whereas activating VTA-mPFC blocks depression even though they both come from the same VTA region.
  • Addiction may be similarly influenced by manipulating these circuits.
  • Speech or swallowing dysfunction may be improved by targeting a specific projection from the periaqueductal gray (PAG) to the nucleus ambiguus (NAmb).
  • PAG periaqueductal gray
  • NAmb nucleus ambiguus
  • Other disorders that may be treated using specific circuits in the brain include but are not limited to obesity and feeding behavior, anxiety, neuroendocrine problems, and the like.
  • compositions comprising, consisting essentially of, or consisting of tire above-described gene transfer vectors and a pharmaceutically acceptable (e.g., physiologically acceptable) carrier.
  • a pharmaceutically acceptable carrier e.g., physiologically acceptable
  • additional components can be included that do not materially affect the composition (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.).
  • the composition consists of the gene transfer vector and the pharmaceutically acceptable carrier, the composition does not comprise any additional components.
  • Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art.
  • compositions can be generated in accordance with conventional techniques described in, e.g., Remington : The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).
  • Suitable formulations for the composition include aqueous and non- aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, 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 multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the carrier is a buffered saline solution.
  • the inventive gene transfer vector is administered in a composition formulated to protect the gene transfer vector from damage prior to administration.
  • the composition can be formulated to reduce loss of the gene transfer vector on devices used to prepare, store, or administer the gene transfer vector, such as glassware, syringes, or needles.
  • the composition can be formulated to decrease the light sensitivity and/or temperature sensitivity of the gene transfer vector.
  • the composition may comprise a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
  • a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
  • Use of such a composition will extend the shelf life of the gene transfer vector, facilitate administration, and increase the efficiency of the inventive method.
  • Formulations for gene transfer vector -containing compositions are further described in, for example, Wright et al., Curr. Opin. Drug Discov.
  • the composition also can be formulated to enhance transduction efficiency.
  • the gene transfer vector can be present in a composition with other therapeutic or biologically-activc agents.
  • 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 gene transfer vector.
  • Immune system stimulators or adjuvants e.g., interleukins, lipopolysaccharide, and double-stranded RNA, can be administered to enhance or modify the immune response.
  • Antibiotics i.e., microbicides and fungicides, can be present to treat existing infection and/or reduce the risk of future infection, such as infection associated with gene transfer procedures.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide- polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled.
  • biodegradable polymers examples include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • a formulation of the present disclosure comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co- polymers thereof, celluloses, polypropylene, polycthylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone),
  • a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co- polymers thereof, celluloses, polypropylene, polycthylenes, polystyrene, polymers of lactic
  • polysaccharides proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.
  • the composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant.
  • Implants see, e.g., U.S. Patent No. 5,443,505
  • devices see, e.g., U.S. Patent No. 4,863,457
  • an implantable device e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition
  • the composition also can be administered in the form of sustained-release formulations (see, e.g., U.S. Patent No.
  • 5,378,475 comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl- tercphthalate (BHET), and/or a polylactic-glycolic acid.
  • a polyphosphoester such as bis-2-hydroxyethyl- tercphthalate (BHET)
  • BHET bis-2-hydroxyethyl- tercphthalate
  • compositions comprising the gene transfer vectors may be intracerebral (including but not limited to intraparenchymal, intraventricular, or intracistemal), intrathecal (including but not limited to lumbar or cistema magna), or systemic, including but not limited to intravenous, or any combination thereof, using devices known in the art. Delivery may also be via surgical implantation of an implanted device.
  • the inventive method comprises administering a“therapeutically effective amount” of the composition comprising the inventive gene transfer vector described herein.
  • A“therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • the therapeutically effective amount may vary according to factors such as pathology, age, sex, and weight of the individual, and the ability of the gene transfer vector to elicit a desired response in the individual.
  • the dose of gene transfer vector in the composition required to achieve a particular therapeutic effect typically is administered in units of vector genome copies per cell (gc/cell) or vector genome copies/per kilogram of body weight (gc/kg).
  • gc/cell vector genome copies per cell
  • gc/kg vector genome copies/per kilogram of body weight
  • the vector is an adenovirus, adeno-associated virus (AAV), retrovirus or lentivirus vector.
  • AAV vector is pseudo typed.
  • the AAV vector is pseudotyped with AAVrh.10, AAV8, AAV9, AAV5, AAVhu.37, AAVhu.20, AAVhu.43, AAVhu.8, AAVhu.2, or AAV7 capsid.
  • the AAV vector is pseudotyped with AAVrh.10, AAV8, or AAV5.
  • the AAV vector is AAV2, AAV5, AAV7, AAV8, AAV9 or AAVrh.10.
  • a dose of the viral vector may be about 1 x 10 11 to about 1 x 10 16 genome copies, about 1 x 10 12 to about 1 x 10 15 genome copies about 1 x 10 11 to about 1 x 10 13 genome copies, CM- about 1 x 10 13 to about 1 x 10 15 genome copies.
  • the composition is administered once to the mammal. It is believed that a single administration of the composition will result in persistent expression in the mammal with minimal side effects.
  • the composition may be administered to the mammal two or more times (e.g., 2, 3, 4, 5, 6, 6, 8, 9, or 10 or more times) during a therapeutic period.
  • the disclosure provides compositions and methods for non-invasive targeting of specific neuronal circuits and for non-invasive regulation of the activity of specific neuronal circuits.
  • at least two gene therapy vectors are employed.
  • one retrograde viral vector is delivered to an organ, brain or spinal cord region of a mammal with a population of axons that come from afferent neurons.
  • the viral vector contains an inactive form of a gene which, when activated, can modulate neuronal function.
  • a second vector is delivered to one brain region of the mammal that sends a limited number of projections to the first target, e.g., organ, brain or spinal cord region with a population of axons that come from afferent neuron.
  • one vector expresses a gene encoding a protein that activates the gene or gene product in the other vector.
  • a retrograde vector expresses Cre recombinase within the nervous system region that projects to the target while the other vector, e.g., which contains a conditionally inactivated gene flanked by lox sites which recombine to activate the gene in the presence of Cre, thereby allowing for expression of the gene in the target site.
  • the second viral vector expresses a gene encoding a protein that activates the gene or gene product in the first viral vector.
  • tire second vector expresses Cre recomb inase, e.g., it is injected or indirectly targeted to the target region within the nervous system, while the retrograde vector, e.g., which is injected or indirectly targeted a region that projects to the target region, contains an inactive gene flanked by lox sites which recombine to activate the gene in the presence of Ore.
  • Cre recomb inase e.g., it is injected or indirectly targeted to the target region within the nervous system
  • the retrograde vector e.g., which is injected or indirectly targeted a region that projects to the target region
  • contains an inactive gene flanked by lox sites which recombine to activate the gene in the presence of Ore.
  • Another example uses a similar approach with the Flp recombinase system.
  • one vector expresses a gene encoding a protein that activates the gene or gene product in the other vector.
  • one vector expresses Cre recombinase within the nervous system region that projects to the first target, while the retrograde vector, e.g., which is injected into the first region, contains an inactive gene flanked by lox sites which recombine to activate the gene in the presence of Cre.
  • Cre recombinase
  • one vector e.g., the first vector which is optionally a retrograde vector, contains a therapeutic gene controlled by the Tet “On” promoter, while the second vector is injected, e.g., systemically or to a targeted region, into, for example, a second brain region, expresses the rTTA transactivator that drives expression from the Tet“On” promoter in the presence of tetracycline.
  • the rTTA transactivator that drives expression from the Tet“On” promoter in the presence of tetracycline.
  • These may be delivered through invasive injection directly into the target organ and/or central nervous system regions, allowing subsequent non- invasive control of neuronal function through expression of genes such as ion channels, G proteins or transcription factors, which respond to exogenous drugs or stimuli to regulate neuronal function.
  • the disclosure also provides for a non-invasive method to target specific circuits in the brain.
  • This method utilizes focal disruption of the blood-brain barrier (BBB) with focused ultrasound, which can transiently open the BBB in regions targeted by the ultrasound when microbubbles, e.g., Optison (GE Healthcare) or Definity (Lantheus Medical Imaging) or another cavitating agent is given intravenously.
  • a vector which is optionally a retrograde vector such as a retrograde AAV vector, containing an inactive form of a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hD3q which is flanked by lox sites, is delivered through focused ultrasound to the substantia nigra.
  • DREADD Designer Receptor Exclusively Activated by Designer Drugs
  • the vector delivers the gene to local substantia nigra neurons and to neurons which project into the substantia nigra, one of which is the putamen.
  • a second AAV serotype e.g., serotype 1
  • this vector contains the gene for Cre under the control of a cytomegalovirus (CMV) promoter with a chicken beta-actin (CBA) enhancer, and the Cre open reading frame is flanked by lox sites.
  • CMV cytomegalovirus
  • CBA chicken beta-actin
  • the Cre When the Cre is expressed in the putaminal neurons, it then recombines the DREADD, which is only contained with putaminal neurons projecting to the substantia nigra, thereby activating the DREADD only in these neurons.
  • the same Cre enzyme also recombines the Cre gene to delete and separate the Cre open reading frame from the promoter, thereby stopping further Cre expression, which is no longer needed.
  • the result is expression of a DREADD only within striatonigral neurons (the specific putamen-substantia nigra neurons), and following administration of the drug clozapine-N-oxide (CNO), the DREADD is activated, thereby activating specifically the nigrostriatal neurons to improve symptoms of, for example, Parkinson’s disease.
  • the gene delivered to conditionally activate the striatonigral neurons includes ion channels which respond to chemicals (chemogenetics), ultrasound (sonogenetics), and/or magnetic fields (magnetogenetics).
  • the vectors are used to conditionally overexpress genes that either prevent or delay the onset of a disease, or silence genes that are associated with an increased risk of developing a disease. For example, overexpression of ApoE2 has protective effects in regard to
  • AD Alzheimer’s disease
  • LDLR Alzheimer’s disease
  • genes associated with an increased risk for a disease can be silenced: for example, ApoE4 or RABIO (Alzheimer’s disease), and PINK1, PARKI/4, or LRRK2 (Parkinson’s disease).
  • a retrograde vector such as a retrograde AAV vector, containing Cre under the control of a cytomegalovirus (CMV) promoter with a chicken beta-actin (CBA) enhancer that is delivered through focused ultrasound to the substantia nigra
  • CMV cytomegalovirus
  • CBA chicken beta-actin
  • a non-retrograde vector having an inactive form of a gene such as a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hD3q which is flanked by lox sites, that is delivered through focused ultrasound to the striatum.
  • the vectors deliver the genes to local neurons in distinct regions, e.g., substantia nigra neurons and to neurons which project into the substantia nigra.
  • a second AAV vector is delivered to the second site with a second session of focused ultrasound.
  • a retrograde vector contains the gene for Cre under the control of a
  • CMV cytomegalovirus
  • CBA chicken beta-actin
  • the Cre When the Cre is delivered to the same cells as those with the conditionally inactivated gene that is flanked by lox sites, it recombines the gene, e.g., DREADD, thereby activating it only in the neurons where the vector having the gene was delivered.
  • the result is expression of a DREADD, e.g., only within striatonigral neurons, and following administration of the drug clozapine-N- oxide (CNO), the DREADD is activated, thereby activating the neurons with DREADD to improve symptoms of, for example, Parkinson’s disease.
  • the conditionally inactivated gene includes ion channels which respond to chemicals (chemogenetics), ultrasound (sonogenetics), and/or magnetic fields (magnetogenetics).
  • Diseases or disorders that may benefit from use of the vectors described herein include but are not limited to Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Adrenoleukodystrophy, Agenesis of the corpus callosum, Agnosia, Aicardi syndrome, Alexander disease, Alpers' disease,
  • Alternating hemiplegia Alzheimer’s disease, Amyotrophic lateral sclerosis (see Motor Neuron Disease), Anencephaly, Angel man syndrome, Angiomatosis, Anoxia, Aphasia, Apraxia, Arachnoid cysts, Arachnoiditis, Amold-Chiari malformation, Arteriovenous malformation, Asperger's syndrome, Ataxia Telangiectasia, Attention Deficit Hyperactivity Disorder, Autism, Auditory processing disorder, Autonomic Dysfunction, , Back Pain, Batten disease, Behcet's disease, Bell's palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bilateral frontoparietal polymicrogyria, Binswanger's disease, Blepharospasm, Bloch-Sulzberger syndrome, Brachial plexus injury, Brain abscess, Brain damage, Brain injury, Brain tumor, Brown-Sequard syndrome, Canavan disease, Carpal tunnel syndrome
  • Leukodystrophy Lewy body dementia, Lissencephaly, Locked-In syndrome,
  • Metachromatic leukodystrophy Microcephaly, Migraine, Miller Fisher syndrome, Mini-Strokes, Mitochondrial Myopathies, Mobius syndrome, Monomelic amyotrophy, Motor Neuron Disease, Motor skills disorder,
  • Moyamoya disease Mucopolysaccharidoses (including the subset referred to as Hurler Syndrome, Hurler-Scheie syndrome, Scheie syndrome, Hunter syndrome, Sanfilippo syndromes A-D, Morquio syndromes A and B, Maroteaus-Lamy syndrome, Sly syndrome, and Natowicz syndrome), Multi-Infarct Dementia, Multifocal motor neuropathy, Multiple sclerosis, Multiple system atrophy with postural hypotension, Muscular dystrophy, Myalgic encephalomyelitis,
  • Myasthenia gravis Myelinoclasdc diffuse sclerosis, Myoclonic Encephalopathy of infants, Myoclonus, Myopathy, Myotubular myopathy, Myotonia
  • Rasmussen's encephalitis Reflex sympathetic dystrophy syndrome, Refsum disease, Repetitive motion disorders, Repetitive stress injury, Restless legs syndrome, Retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, Rombergs-Syndrome, Rabies, Saint Vitus dance, Sandhoff disease,
  • the method for targeting a specific population of mammalian neurons for activatable expression of a gene product includes administering an effective amount of a composition comprising a viral vector with a conditionally activatable gene or gene product, which viral vector is capable of retrograde uptake into neurons to a structure in the mammal to be modulated which contains axons from regulatory neurons, and a composition comprising a second viral vector expressing a gene, the product of which can activate the gene in the first viral vector, which second vector is directly or indirectly targeted to a different region containing a subset of the cell bodies that send the axons to the structure to be modulated.
  • the method for targeting a specific population of mammalian neurons for activatable expression of a gene product includes administering an effective amount of a composition comprising a first viral vector with a conditionally activatable gene or gene product, to a structure in the central nervous system of the mammal to be modulated, and a composition comprising a second viral vector expressing a gene, the product of which can activate the gene in the first viral vector, to a different region in the central nervous system that sends axons to the structure to be modulated, which second viral vector is capable of retrograde uptake into neurons.
  • the method for targeting a specific population of mammalian neurons for activatable expression of a gene product includes administering an effective amount of a composition comprising a first viral vector with a conditionally activatable gene or gene product, to a structure in the central nervous system of the mammal to be modulated, and a composition comprising a second viral vector expressing a gene, the product of which can activate the gene in the first viral vector, wherein one of the vectors is capable of retrograde uptake into neurons.
  • the method for targeting a specific population of mammalian neurons for activatable expression of a gene product includes administering an effective amount of a composition comprising a first viral vector with a conditionally activatable gene or gene product, to a structure in the central nervous system of the mammal to be modulated, and a composition comprising a second viral vector expressing a gene, the product of which can activate the gene in the first viral vector, to the structure.
  • the first vector is systemically administered. In one embodiment, the first vector is locally administered. In one embodiment, the second vector is systemically administered. In one embodiment, the second vector is locally administered. In one embodiment, focused ultrasound is employed fra" targeted delivery of at least one vector. In one embodiment, focused ultrasound is employed for sequential targeted delivery of the first and second vector, e.g., to the same or to different sites. In one embodiment, focused ultrasound is employed for concurrent targeted delivery of the first and second vector, e.g., to the same region. In one embodiment, both vectors are injected directly into the brain into the same region, at the same time (e.g., day) or sequentially, e.g., on the same or different days.
  • both vectors are injected directly into the brain at different sites at tire same time or sequentially, e.g., on tire same or different days.
  • both vectors are targeted to the brain, e.g., after systemic administration and focused ultrasound, into the same region, e.g., sequential administration on different days.
  • both vectors are targeted to the brain, e.g., after systemic administration and focused ultrasound, to different sites, e.g., sequential administration on different days in one embodiment, invasive delivery, e.g., via opening up the skull, is employed for one or both vectors.
  • invasive delivery is employed for one vector and non-invasive delivery, e.g., systemic injection and focused ultrasound, is employed for the other vector to the same or a different site than the vector that is invasively delivered.
  • expression of the conditionally activatable gene is in the brain. In one embodiment, expression of the conditionally activatable gene is in
  • the viral vector comprises an adeno- associated virus, adenovirus, canine adenovirus, herpes simplex virus, or lentivirus vector. In one embodiment, the viral vector comprises a retrograde form of adeno-associated virus, lentivirus or canine adenovirus.
  • the first viral vector contains recognition sites capable of recombination and activation of the gene in the first vector and the second vector encodes a recombinase that is specific for the recognition sites.
  • the recombinase comprises Cre or Flp.
  • the gene product is activatable by an exogenous agent. In one embodiment, the method includes administering the exogenous agent to the mammal.
  • the gene product prevents or inhibits one or more symptoms of a neurological disease or disorder.
  • the recombinase is conditionally activated by an exogenous chemical or stimulus.
  • the gene in the first viral vector does not express a functional protein autonomously
  • the second vector expresses a protein that is capable of activating gene expression or protein function from the gene in the viral vector.
  • the second vector expresses a regulatable or autonomously active transcription factor capable of transactivating expression of the gene product encoded from the viral vector.
  • at least one of the viral vectors is delivered via direct infusion into the structure.
  • at least one of the viral vectors is delivered through focal disruption of the blood-brain barrier using focused ultrasound.
  • m one of the viral vectors is targeted to one brain region in a single treatment, followed by targeting of the other vector to a second brain region in a second treatment. In one embodiment, the two treatments are separated by at least 24 hours.
  • the mammal is a human. In one embodiment, the mammal is a non-human primate. In one embodiment, the mammal is rodent, swine, caprine, ovine, bovine, equine, canine or feline.

Abstract

L'invention concerne des méthodes et des vecteurs pour cibler des régions dans le système nerveux central pour l'administration d'un gène inactivé de manière conditionnelle et d'une recombinase.
EP20758033.3A 2019-08-01 2020-07-31 Thérapie génique ciblée pour traiter des maladies neurologiques Pending EP4007609A1 (fr)

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US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US4863457A (en) 1986-11-24 1989-09-05 Lee David A Drug delivery device
US5378475A (en) 1991-02-21 1995-01-03 University Of Kentucky Research Foundation Sustained release drug delivery devices
US5464758A (en) 1993-06-14 1995-11-07 Gossen; Manfred Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
US5443505A (en) 1993-11-15 1995-08-22 Oculex Pharmaceuticals, Inc. Biocompatible ocular implants
US6342390B1 (en) 1994-11-23 2002-01-29 The United States Of America As Represented By The Secretary Of Health And Human Services Lipid vesicles containing adeno-associated virus rep protein for transgene integration and gene therapy
FR2814642B1 (fr) 2000-10-03 2005-07-01 Ass Pour Le Dev De La Rech En Souris transgenique pour la recombinaison ciblee mediee par la cre-er modifiee
US6723551B2 (en) 2001-11-09 2004-04-20 The United States Of America As Represented By The Department Of Health And Human Services Production of adeno-associated virus in insect cells
MA44546B1 (fr) * 2016-06-15 2021-03-31 Univ California Virus adéno-associés variants et procédés d'utilisation
US20200054711A1 (en) * 2017-02-28 2020-02-20 The Penn State Research Foundation Regenerating functional neurons for treatment of neural injury caused by disruption of blood flow

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