VECTORS AND GENE THERAPY FOR TREATING CORNELIA DE LANGE SYNDROME (2023)

This application claims the benefit of provisional application U.S. Ser. No. 63/196,581, filed on Jun. 3, 2021, which is incorporated herein by reference in its entirety.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 26, 2022, is named 9060_0101-us2_SL.txt and is 19,221 bytes in size.

The present invention relates to AAV gene therapy vectors, AAV replicons, and pharmaceutical compositions for delivering a human HDAC8 gene for treating Cornelia de Lange Syndrome. In addition, methods of treatment and gene transfer are provided.

Cornelia de Lange Syndrome (CdLS) is a genetic disease arising from mutations that impair chromatin structure and function [1, 2]. The clinical syndrome is marked by distinctive physical abnormalities including craniofacial appearance, limb malformations and growth abnormalities. Additionally, patients may have moderate to severe intellectual disability with poor adaptive behavior and sometimes aggression and self-injury [3]. CdLS exhibits a wide range of severity which partially correlates with the responsible gene and specific causative mutation. In addition, some subjects are mosaics with a mixture of affected and unaffected cells [4].

The primary cause of CdLS is a defective chromatin organization with downstream defects in gene expression and DNA replication [5]. Chromatin cohesion is mediated by a multiprotein protein complex called cohesin. The cohesin core is composed of four subunits, the SMC1A, SMC3, RAD21 and STAG proteins, which together form a ring which encloses the DNA. Accessory proteins include NIPLB and MAU which load the cohesin core onto chromatin and HDAC8 which is required for cohesin recycling during the cell cycle. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in prophase and anaphase.

The most common type of CdLS arises from NIPBL mutations which account for 65% of all subjects. Mutations in four other genes, HDAC8, SMC1A, SMC3 and RAD21 account for the remaining cases. The clinical presentation overlaps for mutations in any of these genes. The observed frequency is about 1 in 20,000 births.

The present invention relates to CdLS arising from mutations in histone deacetylase 8 (HDAC8) [6, 7]. This gene is located on the X chromosome and therefore genetic mechanisms are different from other CdLS genes in which patients are usually homozygote or compound heterozygotes. Patients with HDAC8 mutations can be male hemizygotes or female heterozygotes.

The HDAC8 gene encodes a histone deacetylase protein. Histone acetylation/deacetylation alters chromosome structure and affects transcription factor access to DNA. HDAC8 belongs to class I of the histone deacetylase family. It catalyzes the deacetylation of lysine residues in the histone N-terminal tails and represses transcription in large multiprotein complexes with transcriptional co-repressors. But the critical function may be SMC3 deacetylation required for release of the cohesin complex released from chromatin. The gene is expressed in most organs and cell types in a developmentally-controlled fashion but at low levels (2 reads per million mRNA molecules after size adjustment). There are alternate isoforms of the protein generated by alternate RNA splicing but based on exon junction counts the major form is GenBank NM_018486.3 encoding isoform 1, a protein of 377 amino acids (FIG. 1).

The effects of HDAC8 mutations are multiple, and gene expression pattern in many organs is perturbed in CdLS mice [8]. In the case of skull development, global deletion of Hdac8 in mice leads to perinatal lethality due to skull instability, and this is phenocopied by conditional deletion of Hdac8 in cranial neural crest cells [9]. Hdac8 specifically represses the aberrant expression of homeobox transcription factors such as Otx2 and Lhx1. Other transcription factors are misregulated in neurons resulting in cell death which may account for the behavioral and cognitive phenotypes [10]. HDAC8 function may also be perturbed in cancers and this provides a potential molecular target for therapies [11].

A structural model of both wildtype and mutant human HDAC8 proteins has been determined by X-ray crystallography. The active site has been identified and is marked by a catalytic zinc ion [12]. Some mutations, such as C153F mutation, trigger conformational changes that block acetate product release channels, resulting in only 2% residual catalytic activity. In contrast, the H334R mutation causes structural changes in a polypeptide loop distant from the active site and results in 91% residual activity, but the thermostability of this mutant is significantly compromised [12]. Knowledge of the structure of HDAC8 allows the development of small molecules that specifically bind to active site and modify function [11, 13].

The present disclosure provide methods and gene therapy vectors relates for treating CdLS. These vectors are AAV vectors that package an AAV replicon which comprises, in 5′ to 3′ direction, (i) a first AAV inverted terminal repeat (ITR), (ii) a promoter operably linked to an HDAC8 open reading frame, (iii) a polyadenylation (pA) signal operably linked to the HDAC8 open reading frame, and (iv) a second AAV ITR. In an embodiment the, second ITR is the inverse complement of the first ITR. In other embodiments the ITRs can be the flip and fib configuration or any other configuration that produce infectious AAV vectors.

In an embodiment, the ITRs are from AAV serotype 2 or a neurotropic AAV serotype. In an embodiment, the promoter for controlling HDAC8 gene expression is a human HDAC8 promoter or a human EF1a promoter. In an embodiment the HDAC8 open reading frame encodes an HDAC8 protein having an amino acid sequence of FIG. 1 or that of any other isoform of HDAC8. In embodiments, the HDAC8 open reading frame can encode a mutant HDAC8 protein that is therapeutically active. In an embodiment, the pA signal is a human growth hormone pA signal. In some embodiments the AAV replicon comprises one of the nucleic acid sequences in FIGS. 3A-C.

In accordance with the disclosure, the replicon is present in a plasmid used to produce the AAV vectors of the disclosure.

In another aspect, the disclosure relates to recombinant AAV (rAAV) comprising AAV capsid proteins or AAV pseudocapsid proteins and an AAV replicon of the disclosure packaged therein. In an embodiment, the capsids are from AAV serotype 9 or a neurotropic AAV serotype.

A further aspect provides a pharmaceutical composition comprising an rAAV of the disclosure and a pharmaceutically-acceptable carrier.

Further embodiments of the disclosure embrace methods for treating or ameliorating one or more symptoms of CdLS which comprise administering a pharmaceutical composition of the disclosure to a subject in an amount and for a time sufficient to treat or ameliorate the one or more symptoms of CdLS in said subject. In some embodiments, the subject is a rodent or a non-human primate. In some embodiment the subject is a human, including children, teenagers and adults. In embodiments, the composition is administered ICV or IV.

A further aspect provides a method of gene transfer for treating or ameliorating one or more symptoms of CdLS which comprises administering an rAAV of the disclosure to a mammal in an amount and for a time sufficient to treat or ameliorate the one or more symptoms of CdLS in said mammal. In some embodiments, the mammal is a human, a rodent or a non-human primate. In some embodiment the mammal is a human, including children, teenagers and adults. In embodiments, the composition is administered ICV or IV.

FIG. 1 provides the amino acid sequence of the HDAC8 protein (SEQ ID NO: 1), which has 377 amino acids and a mass of 41,758 Da.

FIGS. 2A-C depict schematic diagrams of the AAV CdLS vectors for (A) AAV9-pHDAC8-hHDAC8, (B) AAV9-pEF1a-hHDAC8, and (C) AAV9-pEF1A-hHDAC8-FLAG.

FIGS. 3A-C provide the nucleic acid sequence of the replicon portion of the AAV CdLS vectors for (A) AAV9-pHDAC8-hHDAC8 (SEQ ID NO: 2), (B) AAV9-pEF1a-hHDAC8 (SEQ ID NO: 3), and (C) AAV9-pEF1A-hHDAC8-FLAG (SEQ ID NO: 4).

FIG. 4 is a bar graph showing AAV-encoded human HDAC8 mRNA levels relative to endogenous mouse HDAC8 levels. The average level in the indicated organs is shown (n=3). Black bars: AAV9-pEF1a-hHDAC8; stippled bars: AAV9-pEF1A-hHDAC8-FLAG; cross-hatched bars: AAV9-pHDAC8-hHDAC8. Abbreviation: DRG, dorsal root ganglion.

In order that the present invention may be more readily understood, certain terms are defined below. Additional definitions may be found within the detailed description of the invention.

Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise.

The term “including” is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., cows, pigs), companion animals (e.g., dogs, cats) and rodents (e.g., mice and rats).

The term “non-human mammal” means a mammal which is not a human and includes, but is not limited to, a mouse, rat, rabbit, pig, cow, sheep, goat, dog, non-human primate, or other non-human mammals typically used in research. As used herein, “mammals” includes the foregoing non-human mammals and humans.

As used herein, “treating” or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results. The term may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g., a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The term “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.

As used herein, the terms “preventing” and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition, disease or disorder. Accordingly, “prevention” may refer to prophylactic or preventive measures. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g., a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term “prevention” includes slowing the onset of disease relative to the absence of treatment and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus “preventing” or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition or preventing or delaying the development of symptoms associated with the condition.

As used herein, an “effective amount,” “therapeutically-effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.

As used herein, the term “nucleic acid molecule” is intended to include DNA molecules, RNA molecules (e.g., mRNA, shRNA, siRNA, microRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecules of the invention may be single-, double-, or triple-stranded. A nucleic acid molecule of the present invention may be isolated using sequence information provided herein and well known molecular biological techniques (e.g., as described in Sambrook et al., Eds., MOLECULAR CLONING: A LABORATORY MANUAL 2ND ED., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993).

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, such as viruses. The polynucleotide to be delivered, sometimes referred to as a “target polynucleotide” or “transgene,” may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest). Vector thus includes a biological entity, such as an AAV or other virus, used for the delivery of genes into an organism or introduction of foreign genes into cells.

“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. How cytometry and Western blot, measurement of DNA and RNA by heterologous 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” or “gene transfer” refers to the introduction of an exogenous polynucleotide into a cell for gene therapy, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.

“Gene therapy” 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” 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 e trophic. The term does not necessarily imply any replication capacity of the virus.

The term “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. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention 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. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; 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 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 are envisioned. 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 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. By way of illustration, a transcriptional regulatory sequence (TRS) 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. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, 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 aide 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. In particular, as is well known in numerous molecular biological systems, particular DMA 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 DMA being transcribed. Typical examples of such sequence-specific terminators include polyadenylation (“polyA”) sequences, e.g., SV40 polyA. In addition to or in place of such sequence-specific terminators, insertions of relatively long DMA 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. A variety of such terminator sequences are known in the art; and illustrative uses of such sequences within the context of the present disclosure are provided below.

“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 the 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.

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 elements and a gene or genes to which they are operably linked for egression 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.

The terms “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.

The term “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 By way of a non-limiting example, 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 afferent 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.

The present disclosure provides AAV vectors for use in gene therapy for treating Cornelia de Lange Syndrome (CdLS) associated with mutations in HDAC8. In accordance herewith, the AAV vector is for delivery of AAV replicon comprising, in 5′ to 3′ direction, a first AAV inverted terminal repeat (ITR), a promoter operably linked to an HDAC8 open reading frame, a polyadenylation (pA) signal, and a second AAV ITR.

AAV vectors have many applications in gene therapy for many reasons, including their tropism for specific cell types, their ability to infect both dividing and non-dividing cells, and their ability for genomic integration.

AAV 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 or a herpes virus), or expression of helper genes, for efficient replication. AAV vectors used for administration of therapeutic nucleic acids 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.

AAV vectors can be generated using any AAV serotype known in the art. Several 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): 316-327 (2006)). Generally, 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. In contrast, AAV serotypes 6, 10 (also referred to as Rh10), 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, B. J., Hum. Gene Ther., 16: 541-550 (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: 6823-33 (1997); Srivastava et al., J. Virol., 45: 555-64 (1983); Chiorini et al., J. Virol., 73: 1309-1319 (1999); Rutledge et al., J. Virol., 72: 309-319 (1998); and Wu et al., J. Virol., 74: 8635-47 (2000)).

Generally, the cap proteins, which determine the cellular tropicity of the AAV particle, and related cap protein-encoding sequences, are significantly less conserved than Rep genes across different AAV serotypes. In view of the ability Rep and ITR sequences to cross-complement corresponding sequences of other 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. In contrast, 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. Pat. No. 6,723,551; Flotte, Mol. Ther., 13(1): 1-2 (2006), Gao et al., J. Virol., 78: 6381-6388 (2004), Gao et al., Proc. Natl. Acad. Sci. USA, 99: 11854-11859 (2002), De et al., Mol. Ther., 13: 67-76 (2006), and Gao et al., Mol. Ther., 13: 77-87 (2006).

In embodiments, the transgene in the AAV replicon has the cDNA for a human or other mammalian HDAC8 gene operably linked to a promoter capable of controlling its expression at therapeutic levels. The exact therapeutic level can be determined by those of skill in the art. Expression of the transgene from gene therapy vectors can be driven by any promoter, including strong promoters such as cytomegalovirus (CMV) and chicken beta actin (CBA) that express in all cell types [25]. In preferred embodiments, weaker promoters are used including elongation factor 1A (Ef1a), or phosphoglycerol kinase (PGK), or a native HDAC8 promoter. Since the HDAC8 phenotype results in a neurological phenotype, neuron specific promoters such as methyl CpG-binding protein 2 (MEP229 MEP545), synapsin (SYN1), somatostatin (SST) can be used, potentially reducing toxicity due to ectopic expression of transgene, especially in liver [14, 17, 26]. For example, expression in GABAnergic neurons can also be achieved using cell-specific promoters [26, 27].

In one embodiment, the AAV vector is generated using an AAV that infects humans (e.g., AAV2). Alternatively, 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). In some embodiments, 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: 528-537 (2006). In one embodiment, 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). In a particular embodiment, the AAV vector of the inventive method comprises a capsid protein from AAV9 in which the genome derived from AAV2 is pseudotyped into AAV9 capsid. In another embodiment, AAV10 (also referred to as “AAVrh.10”), which infects rhesus macaques, is pseudotyped with AAV2 ITRs (see, e.g., Watanabe et al., Gene Ther., 17(8): 1042-1051 (2010); and Mao et al., Hum. Gene Therapy, 22: 1525-1535 (2011)).

A common AAV vector production strategy is triple transfection method, which involves co-transfecting the packaging cell line (usually HEK293 T) with the recombinant AAV plasmid containing the gene of interest (GOI), a plasmid containing the essential rep and cap genes, and a third adenovirus-derived helper plasmid supplying genes needed for replication. For large-scale and preclinical AAV packaging services, the AAV particles are purified using IDX gradient ultracentrifugation to remove impurities and empty capsids. In general, methods of producing and purifying AAV vectors using two plasmid and three plasmid systems are known in the art and any such methods can be used to produce the AAV vectors disclosed herein, see, e.g., U.S. Pat. Nos. 6,503,888; 6,632,670; 8,007,780; 8,642,341; 9,051,542; 10,017,746; 10,087,224; 10,093,947; and 10,982,228.

In some embodiments, virions containing a recombinant AAV vector are prepared based on procedures described by KANTOR et al. (Advances in Genetics, vol. 87, 2014, Chapter 2, “Clinical Applications Involving CNS Gene Transfer”); KAPLITT et al. (Lancet 369: 2097-105, 2007); WORGALL et al. (Human Gene Therapy 19:463-474 (2008); LEONE et al., Sci. Transl Med 4: 165ra163 (2012). In an embodiment, the AAV vector suitable for use in the present invention is produced according to the methods described in U.S. Pat. No. 6,342,390. In an alternate embodiment, the AAV vector suitable for use in the present invention is produced according to the methods described in U.S. Pat. No. 6,821,511.

Packaging cell lines include 293 cells which are human embryonic kidney cells modified to contain a small fragment of human adenovirus genome which includes the adenoviral Ela and E1b genes. Another useful packaging cell line is the 293T cell line which contains the SV40 large T antigen gene Both 293 and 293T cells are readily transfected and efficiently package replication deficient AAV vectors given the other adenovirus helper functions (E2a, E4) in the first helper plasmid and AAV replications and capsid functions in the second helper plasmid.

The present disclosure provides AAV gene therapy vectors for treating CdLS. P articular embodiments included methods for treating or ameliorating one or more symptoms of CdLS which comprises administering the an AAV vector of the disclosure or a pharmaceutical composition of the disclosure to a subject in an amount and for a time sufficient to treat or ameliorate the one or more symptoms of CdLS in the subject. In some embodiments the AAV vector or composition is administered by intracerebroventricular or intravenous routes.

In some embodiments, the subject is a rodent or a non-human primate. In some embodiments the subject is a human.

The present disclosure also contemplates a method of gene transfer for treating or ameliorating one or more symptoms of Cornelia de Lange Syndrome (CdLS) which comprises administering an AAV vector or pharmaceutical composition of the disclosure to a mammal in an amount and for a time sufficient to treat or ameliorate the one or more symptoms of CdLS in said mammal. In some embodiments the AAV vector or composition is administered by intracerebroventricular or intravenous routes.

In some embodiments, the mammal is a rodent or a non-human primate. In some embodiments the mammal is a human.

The most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. See, e.g., Remington: The Science and Practice of Pharmacy 21st Ed., Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005.

The present invention further provides pharmaceutical compositions comprising a AAV vector of the disclosure, together with a pharmaceutically acceptable carrier, excipient or vehicle.

Accordingly, the present invention further provides a pharmaceutical composition comprising an AAV vector of the disclosure. Certain embodiments of the pharmaceutical compositions of the invention are described in further detail below.

An AAV vector of the disclosure may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of the vector in a pharmaceutically acceptable carrier.

The therapeutically-effective amount of the AAV vector of the disclosure will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention, and may be confirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person. The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH may be used. pH buffering agents may be phosphate, citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof. The term further encompasses any agents listed in the US Pharmacopeia for use in animals, including humans.

The term “pharmaceutically-acceptable salt” refers to the salt of the compounds. As used herein a pharmaceutically-acceptable salt retains qualitatively a desired biological activity of the parent compound without imparting any undesired effects relative to the compound. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Acid addition salts include salts derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like, or from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Examples of basic salts include salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals such as calcium and magnesium, and ammonium ions ⁺N(R³)₃(R⁴), where R³ and R⁴ independently designate optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted aryl, or optionally substituted heteroaryl, and more specifically, the organic amines, such as N, N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. Other examples of pharmaceutically acceptable salts are described in “Remington's Pharmaceutical Sciences”, 17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more recent editions, and in the Encyclopaedia of Pharmaceutical Technology.

The pharmaceutical compositions can be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen. Compositions may be formulated for any suitable route and means of administration.

Pharmaceutically-acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.

An acceptable route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch). “Parenteral administration” is typically associated with injection at or in communication with the intended site of action, including infraorbital, intraarterial, intracapsular, intracardiac, intracerebroventricular, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.

Pharmaceutical compositions of the disclosure may be administered alone or in combination with one or more other therapeutic or diagnostic agents. A combination therapy may include an AAV vector of the disclosure combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated. Examples of other such agents include, inter alia, an a psychoactive drug, anti-inflammatory or anti-proliferative agent, growth factors, cytokines, an analgesic, a therapeutically-active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates expression of one or more genes, one or more modifiers of signaling pathways and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.

As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, and the like. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on selected route of administration, the AAV vector may be coated in a material or materials intended to protect it from the action of acids and other natural inactivating conditions to which the AAV vector may encounter when administered to a subject by a particular route of administration.

A pharmaceutical composition of the invention also optionally includes a pharmaceutically acceptable antioxidant. Exemplary pharmaceutically acceptable antioxidants are water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyloleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Compositions of the disclosure may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like into the compositions, may also be desirable. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin.

Exemplary pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Such media and reagents for pharmaceutically active substances are known in the art. The pharmaceutical compositions of the disclosure may include any conventional media or agent unless any is incompatible with the AAV vectors of the disclosure. Supplementary active compounds may further be incorporated into the compositions.

Therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art. In certain embodiments, isotonic agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition. Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.

Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as, e.g., sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Sterile injectable solutions may be prepared by incorporating an AAV vector in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains dispersion medium and other ingredients, such as those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of an AAV vector of the disclosure is administered by, e.g., intravenous, cutaneous or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable protein solutions, taking into consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to binding agents, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. A pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending on a variety of factors, including the subject being treated, and the particular mode of administration. In general, it will be an amount of the composition that produces an appropriate therapeutic effect under the particular circumstances. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the particular circumstances of the therapeutic situation, on a case-by-case basis. It is especially advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage when administered to the subject or patient. As used herein, a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention depends on the specific characteristics of the active compound and the particular therapeutic effect(s) to be achieved, taking into consideration and the treatment and sensitivity of any individual patient.

For administration of an AAV vector, the dosage range will generally be from about 2×10¹⁰ to 5×10¹⁵ genome copies or 1×10¹¹ to 1×10¹⁵ of the host body weight. Exemplary dosages may be 3×10¹² genome copies/kg body weight, 1×10¹³ genome copies/kg body weight, 3×10¹³ genome copies/kg body weight, 1×10¹⁴ genome copies/kg body weight or 3×10¹⁴ genome copies/kg body weight or within the range of 1×10¹² to 3×10¹⁴ genome copies/kg. Dosages may be selected and readjusted as required to maximize therapeutic benefit for a particular subject.

AAV vectors may be administered on one or more times. Intervals between single dosages can be, for example, yearly or longer, including 1 year, 2 years, 5 years, or 10 years.

In certain embodiments, two or more AAV vectors may be administered simultaneously or sequentially, in which case the dosage of each administered compound may be adjusted to fall within the ranges described herein.

Actual dosage levels of the AAV vector alone or in combination with one or more other active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing deleterious side effects to the subject or patient. A selected dosage level will depend upon a variety of factors, such as pharmacokinetic factors, including the activity of the particular AAV vector employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.

Administration of a “therapeutically effective dosage” of an AAV vector of the disclosure may result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention or lessening of impairment or disability due to the disease affliction.

The AAV vector or composition of the present disclosure may be administered via one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by the skilled worker, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for AAV vectors and compositions containing such vectors invention include, e.g., intracerebroventricular, intravenous, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intraperitoneal, subcuticular, intraarticular, subcapsular, subarachnoid, epidural and intracisternal magna injection and infusion.

As described elsewhere herein, an AAV vector may be prepared with carriers that will protect it against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compounds or compositions of the invention may be administered with one or more of a variety of medical devices known in the art. For example, in one embodiment, a therapeutic AAV vector composition of the disclosure may be administered with a needleless hypodermic injection device. Examples of well-known implants and modules useful in the present invention are in the art, including e.g., implantable micro-infusion pumps for controlled rate delivery; infusion pumps for delivery at a precise infusion rate; and injection catheters that direct the drug to specific body compartments. These and other such implants, delivery systems, and modules are known to those skilled in the art.

While some embodiments of the invention have been described by way of illustration, it will be apparent that the invention can be put into practice with many modifications, variations and adaptations, and with the use of numerous equivalents or alternative solutions that are within the scope of persons skilled in the art, without departing from the spirit of the invention or exceeding the scope of the claims.

All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The examples presented herein represent certain embodiments of the present invention. However, it is to be understood that these examples are for illustration purposes only and do not intend, nor should any be construed, to be wholly definitive as to conditions and scope of this invention. The examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail.

Three AAV gene therapy vectors were constructed using a three-plasmid system with an AAV serotype 2 replicon-transgene plasmid and two helper plasmids. The three HDAC8 transgene constructs and associated promoters described below were made by total synthesis and cloned into plasmids with the AAV terminal repeats.

AAV-pHDAC8-hHDAC8. The plasmid AAV-pHDAC8-hHDAC8 contains the AAV2 inverted terminal repeats joined to an expression cassette in which expression of the cDNA for human HDAC8 is driven by its native promoter to produce an expression profile resembling that of the endogenous gene. The vector is shown schematically in FIG. 2A and the nucleotide sequence thereof is in FIG. 3A. The genetic elements of this construct are provided in Table 1A.

AAV-pEF1a-hHDAC8. The plasmid AAV-pEF1a-hHDAC8 contains the AAV2 inverted terminal repeats joined to an expression cassette in which expression of the cDNA for human HDAC8 is driven by the promoter of the human EF1a gene to produce a moderate level of gene expression in all cell types. The vector is shown schematically in FIG. 2B and the nucleotide sequence thereof is in FIG. 3B. The genetic elements of this construct are provided in Table 1B.

AAV-pEF1a-hHDAC8-FLAG. The plasmid AAV-pEF1a-hHDAC8-FLAG contains the AAV2 inverted terminal repeats joined to an expression cassette of in which expression of the cDNA for human HDAC8, modified to add a C-terminal FLAG epitope, is driven by the promoter of the human EF1a gene. The vector is shown schematically in FIG. 2C and the nucleotide sequence thereof is in FIG. 3C. The genetic elements of this construct are provided in Table 1C.

AAV Vector Production. Standard methods for preparing AAV vectors were used as described [22-24] using a three plasmid transfection system in 293T cells. These plasmids are the AAV replicon-transgene plasmids encoding the HDAC8 gene; a first AAV helper plasmid encoding the AAV replication protein (rep) and the serotype-specific AAV capsid protein (cap), in this case the AAV9; and a second AAV helper plasmid encoding the adenovirus E2 and E4 proteins. The adenovirus E1 function was provided by the 293T cells.

Briefly, the three plasmids were transfected into the 293T cells using the PolyFect Transfection Reagent (Qiagen) and cells allowed to grow for 48-72 hours. A cell lysate was produced, and cellular DNA removed by DNase1 digest. The lysate was concentrated and loaded onto an iodixanol step gradient. The AAV band was collected and the suspension buffer composition adjusted to phosphate buffered saline by membrane dialysis. Viral titer was determined by qPCR using primer and probe for inverted terminal repeats [22, 24].

Mutant HDAC8 mice are assessed for clinically-relevant phenotypes informative for assessment of potential therapeutics for CdLS. Readily measured in-life parameters with significant difference between wildtype and mutant mice are identified. Parameters that reflect clinically important phenotypes for human subjects with CdLS, for example, memory or learning, are assessed.

To assess phenotypes in Phase 1 of the study, Hdac8^{tm1a(EUCOMM)Wtsi} mice (MGI:4432268; http://www.informatics.jax.org/allele/MGI:4432268) are divided into cohorts as shown in Table 2 and are examined for physical characteristics with tracking of behavior, including two abnormalities that are prominent in hemizygous males and homozygous females: 1) defects in body structure as assessed by DEXA scan and 2) hematological and serum chemistry abnormalities.

For the physical assessments,

• • whole blood is collected for hematology assessment at 4-5 weeks of age (WOA);
  • Body weights and body composition (UltraFocus DEXA) are measured weekly;
  • Cage-side observations are recorded daily; and
  • Nesting behavior is evaluated over a 24 hour-period at 6-8 WOA;
  • additionally, at a pre-determined age, animals are monitored for five consecutive days using PhenoTyper equipment (Noldus) with video tracking system providing data on general activity over the five-day testing period; and
  • Upon meeting the end-point criteria or at 20 WOA, animals are humanely euthanized and necropsy completed, including examination of the following tissues: whole blood, brain, liver, spleen, heart, kidneys, and lungs.

To assess behaviors in Phase 2 of the study, Hdac8^{tm1a(EUCOMM)Wtsi} mice are divided into cohorts as shown in Table 3 and are tracked.

For the behavioral assessment,

• • animals are subjected to neuromuscular/behavioral tests at a single time-point (4 WOA, or other predetermined age), including spontaneous activity measured using Open Field, coordination measured using Erasmus Ladder, rotarod. Y-maze spatial recognition, and isometric force measurement;
  • Body weights are recorded weekly; and
  • animals are humanely euthanized at the end of the study and necropsy completed as needed, including examination of the following tissues: whole blood, brain, liver, spleen, heart, kidneys, and lungs.

Results. This study provides 1) differences between the wildtype and CdLS mouse; 2) information on gender differences for therapeutic studies, and 3) phenotypes useful for therapeutic studies. It further indicates behavioral and neurological pathologies that are relevant to human CdLS subjects.

To establish safety and toxicology of the AAV CdLS vectors produced in Example 1, wildtype mice were divided into cohorts and administered vector or vehicle by the intracerebroventricular (ICV) or intravenous (IV) routes as indicated in Table 4.

Briefly, 3-4 WOA wildtype C57BL/6J mice (Jackson 000664) were injected with a single dose of 3×10¹² vector or of vehicle via unilateral ICV injection or IV injection. Body weights and clinical observations were recorded weekly for all mice. At ten weeks after injection, animals were humanely euthanized, and necropsy performed. Necropsy included collection of brain, spinal cord, heart, liver, lungs, spinal cord and dorsal root ganglion (DRG). All tissues were split in half, with half fixed for histopathology (H&E staining) and the remaining half snap-frozen for gene expression studies. AAV-mediated HDAC8 expression levels were assessed by quantitative RT-PCR and compared to endogenous mouse HDAC8 RNA. The FLAG epitope tagged vector was used to assess cellular distribution of the AAV-encoded HDAC8 by immunohistochemical (IHC) staining using an anti-histidine antibody. H&E stained slides were evaluated by a certified pathologist.

Results. No morbidity or mortality was observed in any cohort. Weekly body weight determinations showed no systematic differences between groups. Some sporadic differences were observed between groups at individual time points but these were not consistent across time points or by groups.

Endogenous mouse mRNA level was assessed using primers specific to mouse HDAC8 and were normalized to GAPDH reference. Endogenous mRNA levels were similar across all tissues examined (brain, spinal cord, DRG, liver lung heart) and administration of AAV expressing human HDAC had no impact on the endogenous mouse HDAC8 mRNA levels.

The AAV vector-encoded mRNA levels were assessed and normalized to the mean endogenous mouse HDAC8 mRNA level (FIG. 4). Human HDAC8 mRNA was not detected in negative control mice treated with vehicle. The highest expression levels were observed in the brain, at approximately 50 times endogenous levels when human HDAC8 expression was controlled by the Ef1a promoter using ICV injection (FIG. 4, black bars). Both the native and epitope tagged version of human HDAZ8 were expressed at comparable levels (FIG. 4, black and stippled bars). By contrast, HDAC8-mediated expression from the HDAC8 promoter was only increased approximately six times endogenous mouse mRNA in brain. Other tissues had lower expression level consistent with the known biodistribution of AAV vectors administered by ICV route in mice, with liver and heart showing high transgene expression. Relative performance of the two different promoters was consistent across all examined tissues.

In sum, ICV injection of AAV vectors expressing human hDAC8 was sufficient to attain levels of AAV-derived HDAC8 mRNA that exceeded the endogenous mouse HDAC8 mRNA in the brain, dorsal root ganglia and liver. These data suggest that a physiological level of HDAC8 expression may be attained by use of either the endogenous HDAC8 promoter or the EF1a promoter.

A CdLS mouse model is used to assess the efficacy of CNS delivery an impact on phenotype of the AAV vectors of Example 1 expressing wildtype HDAC8. The vectors are delivered into Hdac8^{tm1a(EUCOMM)Wtsi} mice as described in Table 5 using the cohorts listed in Table 6.

For the physical assessments,

• • whole blood is collected for hematology assessment at 4-5 WOA;
  • Body weights are measured weekly;
  • Cage-side observations are recorded daily;
  • Body composition is determined (UltraFocus DEXA) on days 28, 42, 56 post AAV injection
  • Nesting behavior is evaluated over a 24 hour-period at 6-8 WOA;
  • Animals are monitored beginning at 7 WOA for five consecutive days using PhenoTyper equipment (Noldus) with video tracking system providing data on general activity over the five-day testing period; and
  • Upon meeting the end-point criteria or at 20 WOA, animals are humanely euthanized, and necropsy completed, including examination of the following tissues: whole blood, brain, liver, spleen, heart, kidneys, and lungs.

For the behavioral assessments,

• • animals are subjected to neuromuscular/behavioral tests at a single time-point (8 WOA, or other predetermined age), including spontaneous activity measured using Open Field, coordination measured using Erasmus Ladder, rotarod, Y-maze spatial recognition, and isometric force measurement; and
  • animals are humanely euthanized 60 days post injection, tissues collected for H & E staining, histopathology, whole blood hematology and CK levels and necropsy, including examination of the following tissues: whole blood, brain, liver, spleen, heart, kidneys, and lungs.

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