Recombinant AAV preparations

Abstract

The present disclosure provides a pharmaceutical composition for express delivery of recombinant adeno-associated virus (rAAV) particles, generally comprising a buffer, a monovalent salt, a polyhydric alcohol, and a triblock copolymer surfactant. The present invention provides compositions comprising recombinant adeno-associated virus (rAAV) and methods of using them. For example, the compositions disclosed herein can be used to deliver rAAV vectors for gene therapy, for example, to the central nervous system. The pharmaceutical composition can have a pH between 7.0 and 7.4, for example, about 7.2.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 234,408, filed August 18, 2021, the entire contents of which are incorporated by reference herein in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated by reference in its entirety. The XML file (created on August 11, 2022) is named ULP_013WO_SL.xml and is 49 kilobytes in size.

[0003] TECHNICAL FIELD OF THEINVENTION The present disclosure relates generally to compositions comprising recombinant adeno-associated viral vectors and methods for their use. [Background technology]

[0004] 2. Background of the Invention Adeno-associated virus (AAV) is a small non-enveloped virus that packages a linear single-stranded DNA genome. AAV vectors can mediate the transfer of heterologous genes, e.g., genes encoding functional proteins for gene therapy. Several recombinant AAV (rAAV) vectors are under development to treat various disorders (e.g., central nervous system disorders). However, delivery of therapeutic agents to the central nervous system is often difficult. For example, in some situations, it has been observed that viral vectors transduce only those cells in the vicinity of the injection site or otherwise exhibit very limited transduction efficiency.

[0005] The present invention addresses this need, for example, by providing compositions for delivery of rAAV to the central nervous system. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present invention provides compositions comprising recombinant adeno-associated virus (rAAV) and their use.For example, the compositions disclosed herein can be used to deliver rAAV vectors for gene therapy, for example, to the central nervous system.

[0007] In one aspect, a) Recombinant adeno-associated virus (rAAV) particles; b) buffer; c) monovalent salt; d) polyhydric alcohols; and e) triblock copolymer surfactants; A pharmaceutical composition comprising:

[0008] In some embodiments, the pharmaceutical composition has a pH between 7.0 and 7.4, for example, about 7.2.

[0009] In some embodiments, the buffer is a phosphate buffer. In some embodiments, phosphate is present in the composition at a concentration between 5 mM and 30 mM, for example, about 10 mM.

[0010] In some embodiments, the buffer is a Tris buffer. In some embodiments, Tris is present in the composition at a concentration between 5 mM and 30 mM, for example, about 10 mM.

[0011] In some embodiments, the polyhydric alcohol is a sugar alcohol, such as a sugar alcohol selected from the group consisting of erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, and xylitol.

[0012] In some embodiments, the sugar alcohol is sorbitol. In some embodiments, sorbitol is present in the composition at a concentration of at least 1% or at least 5%. In some embodiments, sorbitol is present in the composition at a concentration of between 0.5% and 20%, for example, between 5% and 10%. In some embodiments, sorbitol is present in the composition at a concentration of about 5%. In some embodiments, sorbitol is present in the composition at a concentration of about 10%.

[0013] In some embodiments, the triblock copolymer surfactant is a copolymer of ethylene oxide (EO) and propylene oxide (PO), such as a poloxamer. In some embodiments, the poloxamer is poloxamer 188 (Pluronic® F68). In some embodiments, the poloxamer is present in the composition at a concentration between about 0.0001% and about 0.001%. In some embodiments, the poloxamer is present in the composition at a concentration of at least 0.0001%, at least 0.0005%, or at least 0.001%. In some embodiments, the poloxamer is present in the composition at a concentration of about 0.0001%. In some embodiments, the poloxamer is present in the composition at a concentration of about 0.001%.

[0014] In some embodiments, the polyhydric alcohol is sorbitol and the triblock copolymer surfactant is a poloxamer.

[0015] In some embodiments, the monovalent salt is NaCl, KCl, or a combination thereof.

[0016] In some embodiments, NaCl is present in the composition at a concentration between 100 mM and 250 mM, for example, about 130 mM or about 135 mM.

[0017] In some embodiments, the pharmaceutical composition comprises NaCl and KCl.

[0018] In some embodiments, KCl is present in the composition at a concentration between 0.5 mM and 5 mM, for example, about 2 mM.

[0019] In some embodiments, the pharmaceutical composition further comprises one or more divalent salts, such as MgCl 2 , CaCl 2 , or a combination thereof.

[0020] In some embodiments, MgCl2 is present in the composition at a concentration between 0.5 mM and 5 mM, for example, about 1 mM.

[0021] In some embodiments, CaCl2 is present in the composition at a concentration between 0.5 mM and 5 mM, for example, about 1 mM CaCl2.

[0022] In some embodiments, the rAAV particles are 1×10 10 ~2×10 14 It is present in the composition at a concentration of GC / mL.

[0023] In some embodiments, the rAAV particles comprise an AAV capsid derived from AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVhu37, or a variant thereof. In some embodiments, the rAAV particles comprise an AAV capsid derived from AAV9.

[0024] For example, in some embodiments, the pharmaceutical compositions of the present disclosure comprise: a) Recombinant adeno-associated virus (rAAV) particles; b) Phosphate at a concentration between 5 mM and 30 mM c) NaCl at a concentration between 100 mM and 250 mM; d) sorbitol at a concentration between 1% and 10%; and e) poloxamer at a concentration between 0.0001% and 0.001%; wherein the composition has a pH between 7.0 and 7.4.

[0025] In some embodiments, the pharmaceutical compositions of the present disclosure comprise: a) Recombinant adeno-associated virus (rAAV) particles; b) phosphate at a concentration of about 10 mM; c) NaCl at a concentration of about 135 mM; d) sorbitol at a concentration of about 5%; and e) poloxamer at a concentration of about 0.001%; Including, wherein the composition has a pH of about 7.2.

[0026] In some embodiments, the pharmaceutical compositions of the present disclosure comprise: a) Recombinant adeno-associated virus (rAAV) particles; b) phosphate at a concentration of about 10 mM; c) NaCl at a concentration of about 135 mM; d) sorbitol at a concentration of about 10%; and e) poloxamer at a concentration of about 0.001%; Including, wherein the composition has a pH of about 7.2.

[0027] In some embodiments, the pharmaceutical compositions of the present disclosure comprise: a) Recombinant adeno-associated virus (rAAV) particles; b) Tris at a concentration between 5 mM and 30 mM; c) NaCl at a concentration between 100 mM and 250 mM; d) KCl at concentrations between 0.5 mM and 5 mM; e) MgCl2 at concentrations between 0.5 mM and 5 mM; f) CaCl2 at concentrations between 0.5mM and 5mM; d) sorbitol at a concentration between 1% and 10%; and e) poloxamer at a concentration between 0.0001% and 0.001%; Including, Here, the composition has a pH between 7.0 and 7.4.

[0028] In some embodiments, the pharmaceutical compositions of the present disclosure comprise: a) Recombinant adeno-associated virus (rAAV) particles; b) Approximately 10mM Tris; c) Approximately 130mM NaCl; d) Approximately 2mM KCl; e) Approximately 1mM MgCl2; f) Approximately 1mM CaCl2; d) about 5% sorbitol; and e) about 0.001% poloxamer; Including, wherein the composition has a pH of about 7.2.

[0029] In some embodiments, the poloxamer in the pharmaceutical composition of the present disclosure is poloxamer 188 (Pluronic® F68).

[0030] In some embodiments, the rAAV particles comprise an AAV capsid derived from AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVhu37, or a variant thereof. In some embodiments, the rAAV particles comprise an AAV capsid derived from AAV9.

[0031] In some embodiments, the rAAV particle comprises an AAV capsid and a vector genome packaged within the AAV capsid, wherein the vector genome comprises a partial or complete coding sequence of CDKL5, or a functional fragment or variant thereof.

[0032] In some embodiments, the coding sequence of CDKL5 comprises a nucleotide sequence selected from SEQ ID NOs: 12-18 and 19, or a nucleotide sequence that is at least 95% identical to any of SEQ ID NOs: 12-19.

[0033] In some embodiments, the vector genome comprises the nucleotide sequence of SEQ ID NO:20, or a sequence at least 95% identical thereto.

[0034] In some embodiments, the pharmaceutical composition is suitable for intrathecal administration, which includes intraventricular, lumbar, or intracisternal administration.

[0035] In one aspect, a method of delivering rAAV to the central nervous system of a subject is provided, comprising administering to the subject a pharmaceutical composition as disclosed herein. In some embodiments, the subject is a mammal.

[0036] In some embodiments, the administering step comprises administration by intrathecal administration, including intracerebroventricular, lumbar, or intracisternal administration. In some embodiments, the intrathecal administration comprises intracisternal administration. [Brief description of the drawings]

[0037] The present invention may be more fully understood with reference to the following drawings.

[0038] [Figure 1] FIG. 1 is a bar graph showing the calculated number of genome copies (GC) per μg of DNA in various brain regions (frontal cortex ("Ctx"), caudal cortex, cerebellum, and brain stem) from brain tissue administered with the rAAV formulations disclosed herein, as further described in Example 2.

[0039] [Figure 2A-D]Figures 2A-2D show representative images of tissue sections with staining corresponding to RNA expression of the vector genome (as detected by BaseScope®) in mice administered rAAV formulations disclosed herein, as further described in Example 2. Figures 2A, 2B, 2C, and 2D show images from tissue sections of mice in groups 3, 4, 5, and 6, respectively (see Example 2).

[0040] [Figure 2E-H] Figures 2E-2H are pie charts showing corresponding results from quantification of BaseScope signal, indicating transduction efficiency, in the cortex of mice administered the rAAV formulations disclosed herein, as further described in Example 2.

[0041] [Diagram 3] FIG. 3 is a bar graph showing the tissue percentage (in cortical tissue) in which the majority of cells have at least one genome copy in mice administered an rAAV formulation disclosed herein, as further described in Example 2.

[0042] [Figure 4A-D] Figures 4A-4D show representative images of tissue sections with staining corresponding to RNA expression of CDKL5 (as detected by RNAScope®) in mice administered rAAV (in this case, AAV9-SYN-CDKL5) formulations disclosed herein, as further described in Example 2. Figures 4A, 4B, 4C, and 4D show results from mice in groups 3, 4, 5, and 6, respectively (see Example 2).

[0043] [Diagram 5] 5 shows a Western blot stained for CDKL5 protein expression in caudal cortex tissue from mice administered an rAAV formulation disclosed herein (in this case, AAV9-SYN-CDKL5), as further described in Example 2. Staining for GADPH protein expression is shown as a loading control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description of Certain Embodiments of the Invention Unless otherwise noted, technical terms are used according to conventional usage.Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press 1994 (ISBN 0-19-854287-9);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0045] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0046] About: When the term "about" is used before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0047] Adeno-associated virus (AAV): A small, replication-deficient, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and induces a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the host cell genome. These characteristics make AAV an attractive viral vector for gene therapy. There are currently 13 recognized AAV serotypes (AAV1-13).

[0048] Administer / Administer: Providing or giving an agent (e.g., a therapeutic agent (e.g., a recombinant AAV)) to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular or intravenous administration), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

[0049] Between: Unless otherwise noted, when the term "between" is used to refer to a numerical range, the range includes the particular endpoints. For example, the range "between 1 mM and 10 mM" includes 1 mM, 10 mM, and values ​​greater than 1 mM but less than 10 mM.

[0050] Block copolymer: A "block copolymer" refers to a polymer composed of blocks of different polymerized monomers. A "diblock copolymer" has two different blocks, whereas a "triblock copolymer" has three different blocks.

[0051] Coding sequence: "Coding sequence" refers to a nucleotide sequence that, when operably linked to appropriate regulatory sequences, encodes a polypeptide in vitro or in vivo. A coding sequence may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) and 3' untranslated (3'UTR) sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0052] Codon-optimized: A "codon-optimized" nucleic acid refers to a nucleic acid sequence in which the codons have been changed to be optimal for expression in a particular system (e.g., a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in a mammalian cell or a particular mammalian species (e.g., a human cell). Codon optimization does not change the amino acid sequence of the encoded protein.

[0053] Enhancer: A nucleic acid sequence that increases the rate of transcription by increasing the activity of a promoter.

[0054] Intrathecal: As used herein, "intrathecal" administration refers to administration into the intrathecal space that holds cerebrospinal fluid. Examples of intrathecal administration methods include administration via injection into the spinal cord (e.g., lumbar intrathecal), into the ventricles (e.g., intraventricular (ICV)), or into the subarachnoid space (e.g., into the cisterna magna (intracisternal (ICM)).

[0055] Intron: A stretch of DNA within a gene that does not contain any coding information for a protein. Introns are removed before the messenger RNA is translated.

[0056] Inverted terminal repeats (ITR): Symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as replication origins for viral DNA synthesis and are state-of-the-art cis elements for generating AAV integrating vectors.

[0057] Isolated: An "isolated" biological component (e.g., a nucleic acid molecule, a protein, a virus, or a cell) has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and cells) in the cells or tissues of the organism in which it naturally occurs, or in the organism itself. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

[0058] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0059] Polyhydric alcohol: A polyhydric alcohol is an organic compound that has more than one hydroxyl (-OH) group, including dihydric alcohols, which contain two hydroxyl groups, and alcohols with more than two hydroxyl groups.

[0060] Promoter: A region of DNA that directs / initiates the transcription of a nucleic acid (e.g., a gene). A promoter comprises the necessary nucleic acid sequence near the transcription start site. Many promoter sequences are known to those skilled in the art, and furthermore, different promoter sequences can be combined in an artificial nucleic acid molecule. As used herein, gene-specific endogenous promoter refers to the natural promoter element that regulates the expression of an endogenous gene of interest.

[0061] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that has been completely or partially isolated from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparations and subjected to fractionation to remove various components (e.g., proteins, cell debris, and other components) of the original preparation.

[0062] Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not found in nature or that is made by the artificial combination of two otherwise isolated segments of sequence, which can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules (e.g., by genetic engineering techniques).

[0063] "Recombinant AAV" (rAAV) is described, for example, in Wang et al., "Adeno-associated viral vector as a platform for gene therapy delivery." Nat Rev Drug Discov. 2019 May;18(5):358-378 and Samulski et al., "AAV-Mediated gene therapy for Research and Therapeutic Purposes." Annu Rev Virol. 2014 Nov;1(1):427-51. In this disclosure, rAAV is used interchangeably with "rAAV particle."

[0064] Sequence identity: The identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more pronounced when the ortholog protein or cDNA is derived from a more closely related species (e.g., human and mouse sequences) compared to a more distantly related species (e.g., human and C.elegans sequences).

[0065] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970: Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992: and Pearson et al., Meth. Mol. Rio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents detailed considerations of sequence alignment methods and homology calculations.

[0066] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI), and on the Internet, for use with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Further information can be found at the NCBI website.

[0067] Serotype: A group of closely related microorganisms (e.g., viruses) distinguished by a characteristic set of antigens.

[0068] Stuffer sequence: A sequence of nucleotides contained within a larger nucleic acid molecule (e.g., a vector) that is typically used to create a desired spacing between two nucleic acid features (e.g., between a promoter and a coding sequence) or to extend a nucleic acid molecule so that the nucleic acid molecule is of a desired length. Stuffer sequences do not contain protein coding information and may be of unknown / synthetic origin / and / or unrelated to other nucleic acid sequences within the larger nucleic acid molecule.

[0069] Subject: A living multicellular vertebrate organism, a category that includes humans and non-human mammals. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In one embodiment, the human subject is an adult subject, i.e., a human subject over the age of 18. In one embodiment, the human subject is a pediatric subject, i.e., a human subject between the ages of 0 and 18, inclusive. In some embodiments, the subject (e.g., a human subject) has been administered a corticosteroid. In some embodiments, the subject (e.g., a human subject) has been administered an IgG-degrading protease. In some embodiments, the subject (e.g., a human subject) has been administered a corticosteroid and has also been administered an IgG-degrading protease.

[0070] Synthetic: produced by artificial means in a laboratory. For example, synthetic nucleic acids can be chemically synthesized in a laboratory.

[0071] Untranslated region (UTR): A typical mRNA has a sequence upstream and downstream of the coding sequence. Each contains a 5' untranslated region (5' UTR) and a 3' untranslated region (3' UTR) (see Mignone F. et al., (2002) Genome Biol 3:REVIEWS0004).

[0072] Therapeutically effective amount: An amount of a particular pharmaceutical or therapeutic agent (e.g., a recombinant AAV) sufficient to achieve a desired effect in a subject or cell being treated with the agent. The effective amount of the agent depends on several factors, including, but not limited to, the subject or cell being treated and the mode of administration of the therapeutic composition.

[0073] Treating, ameliorating, or preventing a disease: "Treating" refers to a therapeutic intervention that improves a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to a reduction in the number or severity of signs or symptoms of a disease. "Preventing" a disease refers to inhibiting the complete development of a disease.

[0074] Vector: A vector is a nucleic acid molecule that allows the insertion of a foreign nucleic acid without destroying the vector's ability to replicate and / or integrate in a host cell. A vector may contain a nucleic acid sequence that allows it to replicate in a host cell (e.g., an origin of replication). A vector may also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of the inserted gene. In some embodiments herein, the vector is an AAV vector. The term "vector" may also be used in the general sense of an element that carries genetic information or genetic material, for example, to introduce the genetic material into a cell or organism. Those skilled in the art will readily understand the general use of the term "vector" as an element that carries genetic information or genetic material, as well as the more specific use of the term "vector" to refer to a plasmid or vector genome within a rAAV particle.

[0075] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. "Comprising A or B" means including A, or B, or A and B. It should be further understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustration purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification (including explanations of terms) will control, and further, the above materials, methods, and examples are intended to be illustrative only and not limiting.

[0076] I. Pharmaceutical Compositions In many embodiments, provided compositions are in liquid form, for example as a liquid formulation comprising various components as described herein.

[0077] Buffers and pH The compositions provided generally include a buffer / carrier suitable for injection in a human subject. The buffer / carrier should contain components that prevent rAAV from sticking to the injection tubing but do not interfere with rAAV binding activity in vivo. A variety of pH buffering agents may be suitable for use with the compositions of the present disclosure.

[0078] Non-limiting examples of suitable buffering agents include, for example, phosphate and Tris (tris(hydroxymethyl)aminomethane). For example, in some embodiments, the composition includes phosphate, e.g., phosphate is present in the composition at a concentration between 5 mM and 30 mM phosphate. In some embodiments, phosphate is present at a concentration between 5 mM and 29 mM, between 5 mM and 28 mM, between 5 mM and 27 mM, between 5 mM and 26 mM, between 5 mM and 25 mM, between 5 mM and 24 mM, between 5 mM and 23 mM, between 5 mM and 22 mM, between 5 mM and 21 mM, between 5 mM and 20 mM, between 5 mM and 19 mM, between 5 mM and 18 mM. , between 5 mM and 17 mM, between 5 mM and 16 mM, between 5 mM and 15 mM, between 5 mM and 14 mM phosphate, between 5 mM and 13 mM, between 5 mM and 12 mM, between 5 mM and 11 mM, between 5 mM and 10 mM, between 5 mM and 9 mM, between 5 mM and 8 mM, between 5 mM and 7 mM, or between 5 mM and 6 mM. In some embodiments, phosphate is between 6 mM and 30 mM, between 7 mM and 30 mM, between 8 mM and 30 mM, between 9 mM and 30 mM, between 10 mM and 30 mM, between 11 mM and 30 mM, between 12 mM and 30 mM, between 13 mM and 30 mM, between 14 mM and 30 mM, between 15 mM and 30 mM, between 16 mM and 30 mM, between 17 mM and 30 mM. , between 18 mM and 30 mM, between 19 mM and 30 mM, between 20 mM and 30 mM, between 21 mM and 30 mM, between 22 mM and 30 mM, between 23 mM and 30 mM, between 24 mM and 30 mM, between 25 mM and 30 mM, between 26 mM and 30 mM, between 27 mM and 30 mM, between 28 mM and 30 mM, or between 29 mM and 30 mM. In some embodiments, phosphate is present in the composition at a concentration of at least 5 mM, e.g., about 10 mM, about 15 mM, or about 20 mM. In some embodiments, the phosphate is present in the composition at a concentration of about 10 mM.

[0079] In some embodiments, Tris is present in the composition at a concentration of, for example, between 5 mM and 30 mM. In some embodiments, Tris is present at a concentration of between 5 mM and 29 mM, between 5 mM and 28 mM, between 5 mM and 27 mM, between 5 mM and 26 mM, between 5 mM and 25 mM, between 5 mM and 24 mM, between 5 mM and 23 mM, between 5 mM and 22 mM, between 5 mM and 21 mM, between 5 mM and 20 mM, between 5 mM and 19 mM, between 5 mM and 18 mM. , between 5 mM and 17 mM, between 5 mM and 16 mM, between 5 mM and 15 mM, between 5 mM and 14 mM, between 5 mM and 13 mM, between 5 mM and 12 mM, between 5 mM and 11 mM, between 5 mM and 10 mM, between 5 mM and 9 mM Tris, between 5 mM and 8 mM, between 5 mM and 7 mM, or between 5 mM and 6 mM. In some embodiments, Tris is between 6 mM and 30 mM, between 7 mM and 30 mM, between 8 mM and 30 mM, between 9 mM and 30 mM, between 10 mM and 30 mM, between 11 mM and 30 mM, between 12 mM and 30 mM, between 13 mM and 30 mM, between 14 mM and 30 mM, between 15 mM and 30 mM, between 16 mM and 30 mM, between 17 mM and 30 mM, between 18 mM and 30 mM, between 19 mM and 30 mM, between 20 mM and 21 mM, between 22 mM and 23 mM, between 24 mM and 25 mM, between 26 mM and 27 mM, between 28 mM and 29 mM, between 30 mM and 31 mM, between 31 mM and 32 mM, between 32 mM and 33 mM, between 34 mM and 34 mM, between 35 mM and 35 mM, between 36 mM and 36 mM, between 37 mM and 37 mM, between 38 mM and 38 mM, between 39 mM and 40 mM, between 41 mM and 42 mM, between 42 mM and 43 mM, between 44 mM and 45 mM, between 45 mM and 46 mM, between 46 mM and 47 mM, between 48 mM and 49 mM, between 49 mM and 50 mM, between 49 mM and 50 mM, between 51 mM and 52 mM, between 52 mM and 53 mM, between 53 mM and 54 mM, between 54 mM and 55 mM, between 55 mM and 56 mM, between 56 mM and 57 mM, between 57 mM and 58 mM, between 58 mM and 59 mM, between 59 mM and 59 mM, between 60 mM and 61 mM, between In some embodiments, Tris is present in the composition at a concentration of at least 5 mM, e.g., about 10 mM, about 15 mM, or about 20 mM. In some embodiments, Tris is present in the composition at a concentration of about 10 mM.

[0080] The pH of the provided compositions is generally between 6.5 and 8.5, such as between 7.0 and 8.5, between 7.5 and 8, or between 7.0 and 7.4. In some embodiments, the compositions have a pH of about 7.2.

[0081] Polyhydric alcohol Non-limiting examples of suitable polyhydric alcohols include, for example, polyethylene glycol, propylene glycol, and sugar alcohols.

[0082] In certain embodiments, the polyhydric alcohol is a sugar alcohol. In some embodiments, the polyhydric alcohol is a sugar alcohol selected from the group consisting of erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, and xylitol. In some embodiments, the sugar alcohol is sorbitol. In some embodiments, the polyhydric alcohol is a sugar alcohol in its hydrogenated form in mono-(erythritol, xylitol, sorbitol, mannitol), di-(lactitol, isomalt, maltitol), or polysaccharide form.

[0083] In some embodiments, the polyhydric alcohol is present in the formulation in the range of 0.5% to 20% (unless otherwise noted, percentages of polyhydric alcohol in liquid compositions throughout this disclosure refer to weight / volume percentages; 1 g / 100 mL is equal to 1% (w / v)). In some embodiments, the polyhydric alcohol is present in the formulation in the range of 0.5% to 19%, 0.5% to 18%, 0.5% to 17%, 0.5% to 16%, 0.5% to 15%, 0.5% to 14%, 0.5% to 13%, 0.5% to 12%, 0.5% to 11%, 0.5% to 10%, 0.5% to 9%, 0.5% to 8%, 0.5% to 7%, 0.5% to 6%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2%, 0.5% to 1%, 0.5% to 0.9%, 0.5% to 0.8%, 0.5% to 0.7%, or 0.5% to 0.6%. In some embodiments, the polyhydric alcohol is present in the formulation in the range of 0.6% to 20%, 0.7% to 20%, 0.8% to 20%, 0.9% to 20%, 1% to 20%, 2% to 20%, 3% to 20%, 4% to 20%, 5% to 20%, 6% to 20%, 7% to 20%, 8% to 20%, 9% to 20%, 10% to 20%, 11% to 20%, 12% to 20%, 13% to 20%, 14% to 20%, 15% to 20%, 16% to 20%, 17% to 20%, 18% to 20%, or 19% to 20%. In some embodiments, the polyhydric alcohol is present in the formulation in the range of 1% to 10%. In some embodiments, the polyhydric alcohol is present in the formulation at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the polyhydric alcohol is present in the formulation at about 5% or about 10%.

[0084] In some exemplary embodiments, the composition includes sorbitol, hi some embodiments, sorbitol is present in the formulation in the range of 0.5% to 20%. In some embodiments, sorbitol is present in the formulation at a concentration of 0.5% to 19%, 0.5% to 18%, 0.5% to 17%, 0.5% to 16%, 0.5% to 15%, 0.5% to 14%, 0.5% to 13%, 0.5% to 12%, 0.5% to 11%, 0.5% to 10%, 0.5% to 9%, 0.5% to 8%, 0.5% to 7%, 0.5% to 6%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2%, 0.5% to 1%, 0.5% to 0.9%, 0.5% to 0.8%, 0.5% to 0.7%, or 0.5% to 0.6%. In some embodiments, sorbitol is present in the formulation in the range of 0.6%-20%, 0.7%-20%, 0.8%-20%, 0.9%-20%, 1%-20%, 2%-20%, 3%-20%, 4%-20%, 5%-20%, 6%-20%, 7%-20%, 8%-20%, 9%-20%, 10%-20%, 11%-20%, 12%-20%, 13%-20%, 14%-20%, 15%-20%, 16%-20%, 17%-20%, 18%-20%, or 19%-20%. In some embodiments, sorbitol is present in the formulation in the range of 1%-10%. In some embodiments, sorbitol is present in the formulation at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, sorbitol is present in the formulation at about 5% or about 10%.

[0085] Triblock Copolymer Surfactants Non-limiting examples of suitable triblock copolymer surfactants include nonionic triblock copolymer surfactants, such as copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), such as poloxamers. Poloxamers are triblock copolymers composed of a central hydrophobic chain of poly(propylene oxide) (PPO) flanked by two hydrophilic chains of poly(ethylene oxide) (PEO). Poloxamers typically exhibit a molecular weight range of about 1,000 to about 15,000.

[0086] In some embodiments, the triblock copolymer surfactant is a poloxamer, such as poloxamer 188 (Pluronic F68®).

[0087] In some embodiments, the triblock copolymer surfactant (e.g., poloxamer) is present in the composition at a concentration of at least 0.0001%, at least 0.0005%, or at least 0.001% (unless otherwise noted, percentages of triblock copolymer surfactant (e.g., poloxamer) in liquid compositions throughout this disclosure refer to weight / volume percentages; 1 g / 100 mL is equal to 1% (w / v)). In some embodiments, the triblock copolymer surfactant (e.g., poloxamer) is present in the composition at a concentration of 0.0001% to about 0.001%. In some embodiments, the triblock copolymer surfactant (e.g., poloxamer) is between 0.0002% and 0.001%, between 0.0003% and 0.001%, between 0.0004% and 0.001%, between 0.0005% and 0.001%, between 0.0006% and 0.001%, between 0.0007% and 0.001%, between 0.0008% and 0.001%, between 0.000 The composition may be present in a concentration of between 9% and 0.001%, between 0.0001% and 0.0009%, between 0.0001% and 0.0008%, between 0.0001% and 0.0007%, between 0.0001% and 0.0006%, between 0.0001% and 0.0005%, between 0.0001% and 0.0004%, between 0.0001% and 0.0003%, or between 0.0001% and 0.0002%.

[0088] salt The provided compositions may include a physiologically compatible salt or mixture of salts. In some embodiments, any included salt is adjusted to an ionic strength equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt is adjusted to an equivalent ionic concentration.

[0089] For example, the provided compositions can include one or more salts, such as one or more monovalent salts and / or one or more divalent salts. For example, the provided compositions can include a monovalent salt selected from the group consisting of NaCl, KCl, or a combination thereof. For example, in some embodiments, NaCl is between 100 mM and 250 mM, between 100 mM and 240 mM, between 100 mM and 230 mM, between 100 mM and 220 mM, between 100 mM and 210 mM, between 100 mM and 200 mM NaCl, between 100 mM and 190 mM, between 100 mM and 180 mM, between 100 mM and 170 mM, between 100 mM and 160 mM, between 100 mM and 150 mM, between 100 mM and 140 mM, between 100 mM and 150 mM, between 100 mM and 160 mM, between 100 mM and 170 mM, between 100 mM and 160 mM, between 100 mM and 15 ... In one embodiment, the compound is present in the composition at a concentration between 00 mM and 130 mM, between 100 mM and 120 mM, between 100 mM and 110 mM, between 90 mM and 200 mM, between 110 mM and 200 mM, between 120 mM and 200 mM, between 130 mM and 200 mM, between 140 mM and 200 mM, between 150 mM and 200 mM, between 160 mM and 200 mM, between 170 mM and 200 mM, between 180 mM and 200 mM, or between 190 mM and 200 mM.

[0090] In some embodiments, NaCl is present in the composition at a concentration of about 130 mM or about 135 mM.

[0091] In some embodiments, KCl is present in the composition at a concentration between 0.5 mM and 5 mM, between 0.6 mM and 5 mM, between 0.7 mM and 5 mM, between 0.8 mM and 5 mM, between 0.9 mM and 5 mM, between 1 mM and 5 mM, between 1.5 mM and 5 mM, between 2 mM and 5 mM, between 2.5 mM and 5 mM, between 3 mM and 5 mM, between 3.5 mM and 5 mM, between 4 mM and 5 mM, between 4.5 mM and 5 mM, between 0.5 mM and 4.5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 3.5 mM, between 0.5 mM and 3 mM, between 0.5 mM and 2.5 mM, between 0.5 mM and 2 mM, or between 0.5 mM and 1 mM.

[0092] In some embodiments, KCl is present in the composition at a concentration of about 2 mM.

[0093] Non-limiting examples of suitable divalent salts include MgCl2, CaCl2, or combinations thereof. For example, in some embodiments, MgCl2 is present in the composition at a concentration between 0.5 mM and 5 mM, between 0.6 mM and 5 mM, between 0.7 mM and 5 mM, between 0.8 mM and 5 mM, between 0.9 mM and 5 mM, between 1 mM and 5 mM, between 1.5 mM and 5 mM, between 2 mM and 5 mM, between 2.5 mM and 5 mM, between 3 mM and 5 mM, between 3.5 mM and 5 mM, between 4 mM and 5 mM, between 4.5 mM and 5 mM, between 0.5 mM and 4.5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 3.5 mM, between 0.5 mM and 3 mM, between 0.5 mM and 2.5 mM, between 0.5 mM and 2 mM, or between 0.5 mM and 1 mM.

[0094] In some embodiments, MgCl2 is present in the composition at a concentration of about 1 mM.

[0095] In some embodiments, CaCl2 is present in the composition at a concentration between 0.5 mM and 5 mM, between 0.6 mM and 5 mM, between 0.7 mM and 5 mM, between 0.8 mM and 5 mM, between 0.9 mM and 5 mM, between 1 mM and 5 mM, between 1.5 mM and 5 mM, between 2 mM and 5 mM, between 2.5 mM and 5 mM, between 3 mM and 5 mM, between 3.5 mM and 5 mM, between 4 mM and 5 mM, or between 4.5 mM and 5 mM, between 0.5 mM and 4.5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 3.5 mM, between 0.5 mM and 3 mM, between 0.5 mM and 2.5 mM, between 0.5 mM and 2 mM, or between 0.5 mM and 1 mM.

[0096] In some embodiments, CaCl2 is present in the composition at a concentration of about 1 mM.

[0097] Recombinant AAV (rAAV): In this disclosure, compositions and methods of their use are described, e.g., for delivering rAAV to the central nervous system of a subject, e.g., for gene therapy.

[0098] AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear, single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged into the AAV capsid with equal frequency.

[0099] The AAV genome is characterized by two inverted terminal repeats (ITRs) flanking two open reading frames (ORFs). In the AAV2 genome, for example, the first 125 nucleotides of the ITRs are palindromic, which fold back on themselves to maximize base pairing, forming a T-shaped hairpin structure. The other 20 bases of the ITRs (referred to as D-sequences) remain unpaired. The ITRs are cis-acting sequences important for AAV DNA replication; they are the origin of replication and act as a primer for second strand synthesis by DNA polymerase. The double-stranded DNA formed during this synthesis (referred to as replicative monomers) is used for a second round of self-priming replication, forming replicative dimers. These double-stranded intermediates are processed via a strand displacement mechanism, resulting in single-stranded DNA used for packaging and double-stranded DNA used for transcription. Rep binding elements and terminal separation sites (TRSs) are located within the ITRs. These features are used by the viral regulatory protein Rep during AAV replication to process double-stranded intermediates. In addition to their role in AAV replication, the ITRs are also essential for AAV genome packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration (Days and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

[0100] The left ORF of AAV contains the Rep gene, which encodes four proteins - Rep78, Rep68, Rep52 and Rep40. The right ORF contains the Cap gene, which generates three viral capsid proteins (VP1, VP2 and VP3). The AAV capsid contains 60 viral capsid proteins arranged into an icosahedral symmetry. VP1, VP2 and VP3 are present in a 1:1:10 molar ratio (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008).

[0101] AAV is currently one of the most frequently used viruses for gene therapy.Although AAV infects humans and several other primate species, it is not known to cause disease and induces very mild immune response.The gene therapy vector that utilizes AAV can infect both dividing and resting cells, and persists in an extrachromosomal state without being integrated into the genome of host cell.Due to the advantageous characteristics of AAV, the present disclosure contemplates the use of AAV for the recombinant nucleic acid molecule and method disclosed herein.

[0102] AAV has some desirable characteristics of gene therapy vectors, including the ability to bind to, enter, and enter the nucleus of target cells, the ability to be expressed in the nucleus for a long period of time, and low toxicity.However, the size of the AAV genome is small, so the size of the heterologous DNA that can be integrated is limited.To minimize this problem, AAV vectors that do not code for Rep and integration efficiency element (IEE) have been constructed.The ITRs are retained because they are the cis signals required for packaging (Daya and Berns, Clin Microbiol Rev, 21(4):583-593, 2008). Methods for generating rAAV suitable for gene therapy are well known in the art (see, e.g., U.S. Patent Application Publication Nos. 2012 / 0100606; 2012 / 0135515; 2011 / 0229971; and 2013 / 0072548; and Ghosh et al., Gene Ther 13(4):321-329, 2006) and can be utilized with the recombinant nucleic acid molecules and methods disclosed herein.

[0103] In some aspects, the present disclosure provides for the use of a rAAV disclosed herein for the treatment of a disorder (e.g., a disorder affecting the central nervous system), wherein the rAAV comprises an AAV capsid and a vector genome packaged within the AAV capsid. In some embodiments, the rAAV comprises a packaged genome that includes, as operably linked components, in 5' to 3' order: a 5'-ITR, a promoter sequence, a partial or complete coding sequence of a heterologous gene, or a functional fragment or variant thereof, and a 3'-ITR.

[0104] In some embodiments, the recombinant adeno-associated virus (rAAV) particles are 1×10 10 ~4×10 14 Between GC / mL, 1×10 10 ~3×10 14 Between GC / mL, 1×10 10 ~2×10 14 Between GC / mL, 1×1010 ~1×10 14 Between GC / mL, 1×10 11 ~1×10 13 Between GC / mL, 1×10 12~ 6×10 12 Between GC / mL or 1×10 12 ~4×10 12 The composition is present in a concentration of between 100 and 200 GC / mL.

[0105] In some embodiments, the rAAV comprises an AAV capsid and a vector genome packaged within the AAV capsid, where the vector genome comprises: (a) a promoter sequence; and (b) a partial or complete coding sequence for a heterologous gene (e.g., a heterologous gene encoding a therapeutic gene product), or a functional fragment or variant thereof.

[0106] In some embodiments, the packaged vector genome may further comprise a 5'-ITR sequence, an enhancer, an intron, a consensus Kozak sequence, a polyadenylation signal, and / or a 3'-ITR sequence, as described herein. In some embodiments, the recombinant vector may further comprise one or more stuffer nucleic acid sequences. In one embodiment, a stuffer nucleic acid sequence is located between an intron and the partial or complete coding sequence of the heterologous gene.

[0107] In various embodiments described herein, the rAAV comprises an AAV capsid. The AAV capsid can be derived from any one of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10 or hu37 AAV (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVhu37) and more than 100 variants isolated from human and non-human primate tissues.See, for example, Choi et al., 2005, Curr Gene Ther. 5: 299-310, 2005 and Gao et al., 2005, Curr Gene Ther. 5: 285-297.

[0108] In addition to the aforementioned capsids, variant AAV capsids that have been engineered to have one or more beneficial therapeutic properties (e.g., improved targeting to selected tissues, increased ability to evade the immune response, reduced stimulation of neutralizing antibodies, etc.) are included within the scope of the invention. Non-limiting examples of such engineered variant capsids are disclosed in U.S. Patent Nos. 9,506,083, 9,585,971, 9,587,282, 9,611,302, 9,725,485, 9,856,539, 9,909,142, 9,920,097, 10,011,640, 10,081,659, 10,179,176, 10,202,657, 10,214,566, 10,220,570, 10,230,582, 10,240,592, and 10,316,594. Nos. 10,214,785, 10,266,845, 10,294,281, 10,301,648, 10,385,320, and 10,392,632, and in PCT Publication Nos. WO / 2017 / 165859, WO / 2018 / 022905, WO / 2018 / 156654, WO / 2018 / 222503, and WO / 2018 / 226602, the disclosures of which are incorporated herein by reference.

[0109] In certain exemplary embodiments, the rAAV administered according to the present invention comprises an AAV9 capsid. The AAV9 capsid is a self-assembled AAV capsid composed of multiple AAV9 vp proteins. The AAV9 vp proteins are typically expressed as alternative splice variants encoded by the nucleic acid sequence of SEQ ID NO: 11, which encodes the vp1 amino acid sequence of SEQ ID NO: 1 (GenBank Accession: AAS99264), or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical thereto. These splice variants result in different length proteins of SEQ ID NO: 1. As used herein, AAV9 variants include, for example, those described in WO / 2016 / 049230, U.S. Patent No. 8,927,514, U.S. Patent Publication No. 2015 / 0344911, and U.S. Patent No. 8,734,809.

[0110] As shown herein, the rAAV administered according to the present invention may in some embodiments comprise an AAV9 capsid. However, in other embodiments, another AAV capsid is selected. Tissue specificity may be determined by capsid type. The AAV serotype that transduces the appropriate target (e.g., liver, muscle, lung, or CNS) may be selected as the source of capsid for AAV viral vector (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAVrhl64Rl, AAVrhl64R2, AAVrhl8). See, for example, U.S. Patent Publication No. 2007 / 0036760; U.S. Patent Publication No. 2009 / 0197338; and EP1310571. See also WO 2003 / 042397 (AAV7 and other simian AAVs), US Patent Nos. 7,282,199 and 7,790,449 (AAV8). In addition, AAVs not yet discovered, or recombinant AAVs based thereon, may be used as a source of AAV capsids. These documents also describe other AAVs that can be selected to generate AAVs, and are incorporated by reference. In some embodiments, AAV capsids for use in viral vectors can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV capsids or their encoding nucleic acids. In some embodiments, the AAV capsid is a chimera, comprising domains from two or three or four or more of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomers from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the aforementioned capsids.

[0111] In some embodiments, the rAAV comprises an AAV capsid and a vector genome packaged within the AAV capsid, wherein the vector genome comprises a partial or complete coding sequence for CDKL5, or a functional fragment or variant thereof.

[0112] In some embodiments, the coding sequence of CDKL5 comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12-18 and 19, or a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to any of SEQ ID NOs: 12-19.

[0113] In exemplary embodiments, the rAAV comprises an AAV capsid and a vector genome packaged within the AAV capsid, wherein the vector genome comprises the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical thereto. In some such embodiments, the AAV capsid is an AAV9 capsid.

[0114] Inverted terminal repeat (ITR): In some embodiments, rAAV contains a packaged vector genome that includes AAV ITR sequences, which function as both a vector DNA origin of replication and a packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. In addition, the ITRs serve as targets for single-stranded endonucleatic nicking by the large Rep protein to separate individual genomes from replicative intermediates.

[0115] In some embodiments, the 5'-ITR sequence is derived from AAV2. In some embodiments, the 3'-ITR sequence is derived from AAV2. In some embodiments, the 5'-ITR sequence and the 3'-ITR sequence are derived from AAV2. In some embodiments, the 5'-ITR sequence and / or the 3'-ITR sequence are derived from AAV2 and comprise or consist of SEQ ID NO:2. In other embodiments, the 5'-ITR sequence and / or the 3'-ITR sequence are derived from a non-AAV2 source.

[0116] promoter In various aspects described herein, the rAAV comprises a packaged vector genome that includes a promoter sequence that helps drive and regulate the expression of a heterologous gene. In exemplary embodiments, the promoter sequence is located between the 5'-ITR sequence and the partial or complete coding sequence of the heterologous gene. In some embodiments, the promoter sequence is located downstream of an enhancer sequence. In some embodiments, the promoter sequence is located upstream of an intron sequence.

[0117] In some embodiments, the promoter is a neuron-specific promoter.In one embodiment, the neuron-specific promoter is selected from human synapsin 1 (SYN1) promoter, mouse calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, rat tubulin αI (Ta1) promoter, rat neuron-specific enolase (NSE) promoter, human neuron-specific enolase (ENO2) promoter, human platelet-derived growth factor β chain (PDGF) promoter, human BM88 promoter, and neuronal nicotinic receptor β2 (CHRNB2) promoter.

[0118] In an exemplary embodiment, the neuron-specific promoter is a SYN1 promoter (e.g., a human SYN1 promoter). In one embodiment, the SYN1 promoter (e.g., a human SYN1 promoter) is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO:3. In an exemplary embodiment, the SYN1 promoter (e.g., a human SYN1 promoter) comprises or consists of SEQ ID NO:3.

[0119] In some embodiments, the promoter is selected from a chicken beta-actin (CBA) promoter, a cytomegalovirus (CMV) immediate early gene promoter, a transthyretin (TTR) promoter, a thyroxine-binding globulin (TBG) promoter, and an alpha-1 antitrypsin (A1AT) promoter.

[0120] In an exemplary embodiment, the promoter is a CBA promoter. In one embodiment, the CBA promoter comprises or consists of SEQ ID NO:4.

[0121] In some embodiments, the promoter is a gene-specific endogenous promoter, hi one embodiment, the promoter comprises the native gene promoter elements.

[0122] Other packaged vector genome elements In addition to the promoter and coding sequence of the heterologous gene, the packaged genome may contain other appropriate transcription initiation, termination, enhancer, and efficient RNA processing signals. As described in more detail below, such sequences include splicing and polyadenylation (polyA) signals, regulatory elements that enhance expression, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), and sequences that enhance protein stability.

[0123] In some embodiments, the rAAV comprises a packaged vector genome comprising one or more enhancer sequences. In one embodiment, the enhancer is selected from a cytomegalovirus immediate early gene (CMV) enhancer, a transthyretin enhancer (enTTR), a chicken beta-actin (CBA) enhancer, an En34 enhancer, and an ApoE enhancer. In an exemplary embodiment, the enhancer is a CMV enhancer (e.g., a CMV immediate early gene enhancer). In one embodiment, the CMV enhancer (e.g., a CMV immediate early gene enhancer) comprises or consists of SEQ ID NO:7.

[0124] In some embodiments, the rAAV comprises a packaged vector genome that includes one or more intron sequences. In one embodiment, the intron is selected from the SV40 Small T intron, rabbit hemoglobin subunit β (rHBB) intron, human β-globin IVS2 intron, β-globin / IgG chimeric intron, and hFIX intron. In one exemplary embodiment, the intron is the SV40 Small T intron. In one embodiment, the SV40 Small T intron sequence comprises or consists of SEQ ID NO:8.

[0125] In some embodiments, the rAAV comprises a packaged vector genome that includes a consensus Kozak sequence. In some embodiments, the consensus Kozak sequence is located downstream of an intron sequence. In one embodiment, the consensus Kozak sequence is GCCGCCACC.

[0126] In some embodiments, the rAAV comprises a packaged vector genome comprising a polyadenylation signal sequence. In one embodiment, the polyadenylation signal sequence is selected from the bovine growth hormone (BGH) polyadenylation signal sequence, the SV40 polyadenylation signal sequence, the rabbit β-globin polyadenylation signal sequence, and a gene-specific endogenous polyadenylation signal sequence. In an exemplary embodiment, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence. In one embodiment, the SV40 polyadenylation signal sequence comprises or consists of SEQ ID NO:5.

[0127] II. Methods of Delivering rAAV In one aspect, the disclosure provides a method of delivering rAAV to the central nervous system of a subject, the method comprising administering to the subject a pharmaceutical composition as described herein.

[0128] In exemplary embodiments, the pharmaceutical composition is administered intrathecally, e.g., via intraventricular, lumbar, or intracisternal administration. In some embodiments, the pharmaceutical composition is administered by intracisternal administration.

[0129] For example, in some embodiments, the pharmaceutical composition is administered using an automated intrathecal injector. For example, an injector that influences CSF dynamics, physiological pulsatility, and volume displacement can be utilized to deliver rAAV intrathecally. An example of one possible injector for use in the methods of the present invention is the Pulsar, developed by Alcyone Lifesciences, Inc. TM It is a Smart Intrathecal Delivery Platform.

[0130] The specific dose administered is a uniform dose for each patient, e.g., 1.0 x 10 11 ~1.0×10 14 The dose may be a genome copy (GC) of the virus. Alternatively, the patient's dose may be adjusted to the approximate weight or surface area of ​​the patient. Other factors in determining the appropriate dose may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. Further refinement of the calculations required to determine the appropriate dose for treatment is routinely performed by those skilled in the art, especially in light of the dosage information and assays disclosed herein. The dosage may also be determined through the use of known assays to determine the dosage to be used with appropriate dose-response data. The dosage of an individual patient may also be adjusted as the progression of the disease is monitored.

[0131] In some embodiments, the pharmaceutical composition has a potency of, for example, about 1.0×10 as measured by qPCR or droplet digital PCR (ddPCR). 11 Genome copies / kg patient weight (GC / kg) ~ approx. 1 x 10 14 GC / kg, approx. 5×10 11 Genome copies / kg patient weight (GC / kg) ~ approx. 5 × 10 13 GC / kg, or approximately 1 x 10 12 ~Approx. 1×10 13In some embodiments, the rAAV is administered at a dose of about 1×10 GC / kg of rAAV. 12 ~Approx. 1×10 13 In some embodiments, the rAAV is administered at a dose of about 1.1 x 10 genome copies (GC) / kg. 11 , about 1.3×10 11 , about 1.6×10 11 , about 1.9×10 11 , about 2×10 11 , about 2.5×10 11 , about 3.0×10 11 , about 3.5×10 11 , about 4.0×10 11 , about 4.5×10 11 , about 5.0×10 11 , about 5.5×10 11 , about 6.0×10 11 , about 6.5×10 11 , about 7.0×10 11 , about 7.5×10 11 , about 8.0×10 11 , about 8.5×10 11 , about 9.0×10 11 , about 9.5×10 11 , about 1.0×10 12 , about 1.5×10 12 , about 2.0×10 12 , about 2.5×10 12 , about 3.0×10 12 , about 3.5×10 12 , about 4.0×10 12 , about 4.5×10 12 , about 5.0×10 12 , about 5.5×10 12 , about 6.0×10 12 , about 6.5×10 12 , about 7.0×10 12 , about 7.5×10 12 , about 8.0×10 12 , about 8.5×10 12 , about 9.0×10 12 , about 9.5×10 12 , about 1.0×10 13 , about 1.5×10 13 , about 2.0×10 13 , about 2.5×10 13, about 3.0×10 13 , about 3.5×10 13 , about 4.0×10 13 , about 4.5×10 13 , about 5.0×10 13 , about 5.5×10 13 , about 6.0×10 13 , about 6.5×10 13 , about 7.0×10 13 , about 7.5×10 13 , about 8.0×10 13 , about 8.5×10 13 , about 9.0×10 13 , about 9.5×10 13 The rAAV is administered in a dose of genome copies (GC) / kg. The rAAV can be administered in a single dose or in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses) if required for the desired therapeutic outcome.

[0132] In some embodiments, the methods of delivering rAAV according to the present invention may further comprise administration of an IgG-degrading protease prior to administering the pharmaceutical composition described herein.

[0133] In some embodiments, a method of delivery according to the invention is carried out in a subject (eg, a mammal, such as a human) that has been administered an IgG-degrading protease.

[0134] Examples of proteases that may be used in the present invention include, but are not limited to, those described in, for example, WO / 2020 / 016318 and / or WO / 2020 / 159970 (e.g., cysteine ​​proteases derived from Streptococcus pyogenes, Streptococcus equi, Mycoplasma canis, Streptococcus agalactiae, Streptococcus pseudoporcinus, or Pseudomonas putida).

[0135] In certain embodiments, the IgG-degrading protease is IdeS (SEQ ID NO:9) from Streptococcus pyogenes, or a protease that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In some embodiments, the protease is an engineered variant of SEQ ID NO:9. Examples of engineered IdeS proteases are described in WO / 2020 / 016318 and U.S. Patent Publication Nos. 20180023070 and 20180037962. In some embodiments, the engineered IdeS variant may have one, two, three, four, five, or more amino acid modifications relative to SEQ ID NO:9.

[0136] In certain embodiments, the IgG-degrading protease is IdeZ from Streptococcus equi (SEQ ID NO: 10) or a protease that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the protease is an engineered variant of SEQ ID NO: 10. Examples of engineered IdeZ proteases are described in WO / 2020 / 016318. In some embodiments, the engineered IdeZ variant may have one, two, three, four, five, or more amino acid modifications relative to SEQ ID NO: 10.

[0137] Other proteases that may be used in the present invention include, but are not limited to, the IgdE enzymes from Streptococcus suis, Streptococcus porcinus, and Streptococcus equi, e.g., as described in WO / 2017 / 134274.

[0138] In some embodiments, the IgG-degrading protease may be encapsulated in or complexed with a liposome, nanoparticle, lipid nanoparticle (LNP), polymer, microparticle, microcapsule, micelle, or extracellular vesicle.

[0139] Corticosteroids In some embodiments, the methods provided include administering a composition comprising an rAAV as disclosed herein to a subject who has previously been administered a corticosteroid. The compositions disclosed herein can be administered intrathecally, e.g., intracerebroventricularly, or via intracisternal delivery.

[0140] In various embodiments according to this aspect, the corticosteroid may be selected from prednisolone, prednisone, dexamethasone, hydrocortisone, triamcinolone, methylprednisolone, budesonide, betamethasone, and deflazacort. In an exemplary embodiment, the corticosteroid is prednisolone.

[0141] In various embodiments according to this aspect, the corticosteroid is administered to the subject at least about 12 hours prior to administration of the composition comprising rAAV. In another embodiment, the corticosteroid is administered to the subject at least about 24 hours prior to administration of the composition comprising rAAV. In yet another embodiment, the corticosteroid is administered to the subject at least about 2 days prior to administration of the composition comprising rAAV. In yet another embodiment, the corticosteroid is administered to the subject at least about 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days prior to administration of the composition comprising rAAV. In yet another embodiment, the corticosteroid is administered to the subject at least about 7 days, 14 days, 21 days, or more than 21 days prior to administration of the composition comprising rAAV. In yet another embodiment, the corticosteroid is administered to the subject at least about 1 month, at least about 2 months, or at least about 3 months prior to administration of the composition comprising rAAV, hi some embodiments, the corticosteroid is administered between 12 hours and 4 months, e.g., between 24 hours and 3 months, prior to administration of the composition comprising rAAV.

[0142] In one embodiment, the corticosteroid is administered once prior to administering a composition comprising the rAAV. In another embodiment, the corticosteroid is administered twice prior to administering a composition comprising the rAAV. In yet another embodiment, the corticosteroid is administered three, four, five or more times prior to administering a composition comprising the rAAV.

[0143] Administration of the corticosteroid to a human subject can be by any route, including, but not limited to, oral, intravenous, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via aerosol), buccal (e.g., sublingual), topical (i.e., both the skin and mucosal surfaces, including airway surfaces), intrathecal, intraarticular, intrapleural, intracerebral, intraarterial, intraperitoneal, or intranasal administration. In an exemplary embodiment, the corticosteroid is administered orally.

[0144] In certain embodiments, the dose of the corticosteroid is measured in mg / kg of subject body weight. In other embodiments, the dose of the corticosteroid is measured in mg per dose administered to the subject. Any measurements of doses and dosage units that may be used with the compositions and methods of the present invention may be converted by standard means in the art.

[0145] In certain embodiments, the corticosteroid may be administered in a dose of about 1 mg to about 1000 mg. In some embodiments, the corticosteroid is administered in a dose of about 3 mg to about 300 mg. In some embodiments, the corticosteroid is administered in a dose of about 5 mg to about 150 mg. In some embodiments, the corticosteroid is administered in a dose of about 10 mg to about 100 mg. In some embodiments, the corticosteroid is administered in a dose of about 15 mg to about 80 mg. In some embodiments, the corticosteroid is administered in a dose of about 20 mg to about 60 mg.

[0146] In certain embodiments, the corticosteroid is about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, It may be administered in a dose of about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg.

[0147] In certain embodiments, the corticosteroid may be administered at a dose of about 0.1 mg / kg to about 100 mg / kg of subject body weight. In some embodiments, the anti-CD19 antibody is administered at a dose of about 0.2 mg / kg to about 10 mg / kg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 0.5 mg / kg to about 5 mg / kg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, or about 10 mg / kg of the subject's body weight.

[0148] In some embodiments, the corticosteroid may be administered for a total of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or more days prior to administration of the composition comprising rAAV. In some embodiments, the corticosteroid is administered for between 1 day and 14 days, e.g., between 1 day and 12 days, or between 1 day and 10 days, prior to administration of the composition comprising rAAV. For example, in certain exemplary embodiments, the corticosteroid may be administered at 1 mg / kg / day for 5 days prior to administration of the composition comprising rAAV.

[0149] In some embodiments, the corticosteroid may be administered at 1 mg / kg / day for four weeks, with the first administration occurring five days prior to administration of the composition comprising rAAV. In some embodiments, the corticosteroid may be administered at 1 mg / kg / day for four weeks, with the first administration occurring five days prior to administration of the composition comprising rAAV, followed by a corticosteroid taper over an additional four weeks.

[0150] The method according to this aspect can be used to treat any CNS disorder for which gene therapy may be appropriate. In some embodiments, the CNS disorder is selected from CDKL5 deficiency disorder (CDD), Angelman syndrome, Batten disease, Krabbe disease, Parkinson's disease, Alzheimer's disease, spinal muscular atrophy (SMA) type I, II, III, and IV, X-linked myotubular myopathy, Friedreich's ataxia, Canavan disease, amyotrophic lateral sclerosis (ALS), adrenoleukodystrophy, Huntington's disease, Rett syndrome, and spinocerebellar ataxia.

[0151] Throughout the detailed description, when compositions are described as having, including, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0152] In this disclosure, when an element or component is referred to as being included in and / or selected from a list of described elements or components, it is to be understood that the element or component can be any one of the described elements or components, or that the element or component can be selected from a group consisting of two or more of the described elements or components.

[0153] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether express or implied herein, can be combined in various ways without departing from the spirit and scope of the present invention.For example, when a reference is made to a particular compound, that compound can be used in various embodiments of the compositions of the present invention and / or in the methods of the present invention, unless otherwise understood from the context.In other words, in this disclosure, the embodiments are described and shown in a manner that allows a clear and concise disclosure to be written and drawn, but it is intended and recognized that the embodiments can be combined or separated in various ways without being separated from the present teachings and the present invention.For example, it is recognized that all features described and shown herein can be applicable to all aspects of the invention described and shown herein.

[0154] The phrase "at least one of" should be understood to include each of the listed objects that follow the phrase, and two or more various embodiments of that listed object, individually, unless otherwise understood from context and use. In the context of three or more listed objects, the phrase "and / or" should be understood to have the same meaning, unless otherwise understood from context.

[0155] Use of the terms "include," "includes," "including," "have," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should generally be understood to be open-ended and non-limiting, e.g., not excluding additional, unrecited elements or steps unless otherwise specifically stated or understood from the context.

[0156] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be considered simultaneous.

[0157] The use of any and all examples or exemplary language, e.g., "such as" or "including," herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. EXAMPLES

[0158] The disclosure generally described herein will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the disclosure, and are not intended to limit the scope of the disclosure in any way.

[0159] Example 1: Generation of rAAV compositions This example describes various rAAV-containing compositions used in other examples in this disclosure.

[0160] rAAV expressing heterologous genes were generated in human embryonic kidney (HEK) or HeLa cell producer lines. rAAV compositions were made using the following formulations: [Table 1]

[0161] Example 2: Biodistribution and transduction efficiency of rAAV-containing formulations This example demonstrates the successful delivery of recombinant adeno-associated virus (rAAV) expressing a therapeutic gene product to various brain regions using the compositions disclosed herein.

[0162] In this example, a rAAV expressing CDKL5 (AAV9-SYN-CDKL5) was generated and delivered to mice deficient in CDKL5.

[0163] CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene that can manifest in a wide range of clinical symptoms and severity. The CDKL5 gene encodes the cyclin-dependent kinase-like 5 (CDKL5) protein, which is essential for normal brain development and function. CDD is caused by pathogenic variants in the CDKL5 gene, including deletions, truncations, splice variants, and missense mutations. These variants can reduce the amount of functional CDKL5 protein and / or decrease its activity in neurons.

[0164] Compositions were made that included HeLa-generated AAV9-SYN-CDKL5 and additional components as shown in Table 1 of Example 1. CDKL5-deficient (Cdkl5 KO) mice between 3 and 5 weeks of age were administered 10 μL of either control or rAAV compositions by intracerebroventricular (ICV) injection as shown in Table 2. "Control" groups were administered either saline (Group 2) or a control composition including HEK-generated AAV9-SYN-CDKL5 (Group). Wild-type C57BL / 6 mice were also used as controls (Group 1). [Table 2]

[0165] Mice were sacrificed two weeks after injection and tissues were analyzed for the presence of vector genome (e.g., by quantitative polymerase chain reaction (qPCR)), vector RNA (e.g., using the BaseScope® Assay), CDKL5 RNA (e.g., using the RNAScope® Assay) or CDKL5 protein (e.g., by Western blot).

[0166] FIG. 1 shows the calculated numbers of genome copies (GC) per μg of DNA in various brain regions (frontal cortex, caudal cortex, cerebellum, and brainstem). 6 or greater) of vector genomes (GC / μg DNA) were observed in all brain regions assessed from the brains of rAAV-administered mice.

[0167] 2A-2D show representative images of tissue sections with staining (as detected by BaseScope®) corresponding to RNA expression of vector genomes in mice administered rAAV in Groups 3 (FIG. 2A), 4 (FIG. 2B), 5 (FIG. 2C), and 6 (FIG. 2D). FIGS. 2E-2H are pie charts showing corresponding results from quantification of BaseScope signal (indicative of transduction efficiency) in the cortex of mice administered control or rAAV formulations from Groups 3 (FIG. 2E), 4 (FIG. 2F), 5 (FIG. 2G), and 6 (FIG. 2H). Each pie chart is labeled at the top left with the number of the mouse group for the experiment. FIG. 2E shows the pie chart for mice Group 3 administered with a control composition. FIGS. 2F, 2G, and 2H show the pie charts for Groups 4, 5, and 6, respectively, administered with rAAV formulations disclosed herein. The legend for each pie chart is to the right of each pie chart and indicates numbers from 0 to 4 to represent the amount of genome copies (GC) / cell quantified. Number 4 (fine stippled pattern) represents >10 GC / cell; number 3 (striped pattern) represents 4-10 GC / cell; number 2 (medium stippled pattern) represents 2-3 GC / cell; number 1 (mesh pattern) represents 1 GC / cell; number 0 (open) represents no GC / cell. As shown in Figures 2F, 2G, and 2H (corresponding to results from Groups 4, 5, and 6, respectively), administration of rAAV using the formulations disclosed herein resulted in delivery of >10 GC / cell in a significant percentage of cells in the cortex.

[0168] 3 is a bar graph showing the percentage of regions in which the majority of cells have at least one genomic copy. In groups 4, 5, and 6, this percentage was at least 80%.

[0169] Figures 4A-4D show representative images of tissue sections with staining corresponding to CDKL5 RNA expression (as detected by RNAScope®) in mice administered rAAV in Groups 3 (Figure 4A), 4 (Figure 4B), 5 (Figure 4C), and 6 (Figure 4D). Figure 5 shows a Western blot stained for CDKL5 protein expression in caudal cortical tissue. Staining for GADPH protein expression is shown as a loading control.

[0170] The results described in this example demonstrate that the compositions disclosed herein can be used to deliver recombinant rAAV to various brain tissues and express heterologous genes in the brain.

[0171] Example 3: Delivery of rAAV to the central nervous system by intracisternal or lumbar intrathecal injection This example describes experiments that can be used to evaluate rAAV delivery to the central nervous system using the formulations described herein administered via the intracisternal or lumbar intrathecal routes.

[0172] A formulation containing a rAAV vector encoding a reporter gene (e.g., GFP) can be generated as described in Example 1. A fixed volume of the formulation can be administered to a non-human primate (e.g., a cynomolgus monkey) by injection into the cisterna magna or by lumbar intrathecal injection. During administration, the non-human primate can be in the Trendelenburg position.

[0173] The biodistribution of the rAAV can be evaluated, for example, by analyzing the amount of genome copies (GC / μg DNA) in various tissues.Delivery to the central nervous system can be evaluated by analyzing the amount of GC / μg DNA in various brain tissues.A substantial amount of genome copies across various brain tissues indicates successful delivery to the central nervous system.

[0174] Expression of the reporter gene in various brain tissues and / or at the cellular level can be assessed in tissue sections, for example, using staining or other visualization methods appropriate for the reporter gene.

[0175] Example 4: Delivery of therapeutic rAAV via intracisternal injection rAAV can be produced that expresses genes that code for proteins associated with central nervous system disorders. Compositions that include the rAAV can be similarly formulated as described in Example 1. The composition can be delivered to a subject in need thereof (e.g., a subject that has been diagnosed with a central nervous system disorder or identified as being at risk of developing a central nervous system disorder) via intracisternal injection. The outcome of treatment, improvement, or prevention can be measured in the subject by a method appropriate for the relevant central nervous system disorder.

[0176] Example 5: Delivery of therapeutic rAAV via intra-lumbar IT injection rAAV can be produced that expresses genes that code for proteins associated with central nervous system disorders. Compositions that include the rAAV can be similarly formulated as described in Example 1. The composition can be delivered to a subject in need thereof (e.g., a subject that has been diagnosed with a central nervous system disorder or identified as being at risk of developing a central nervous system disorder) via lumbar IT injection. The outcome of treatment, improvement, or prevention can be measured in the subject by a method appropriate for the relevant central nervous system disorder.

[0177] Example 6: Delivery of therapeutic rAAV via intraventricular injection rAAV can be produced that expresses genes that code for proteins associated with central nervous system disorders. Compositions that include the rAAV can be similarly formulated as described in Example 1. The composition can be delivered to a subject in need thereof (e.g., a subject that has been diagnosed with a central nervous system disorder or identified as being at risk of developing a central nervous system disorder) via intraventricular injection. The outcome of treatment, improvement, or prevention can be measured in the subject by a method appropriate for the relevant central nervous system disorder.

[0178] Incorporation by Reference The entire disclosures of each of the patent documents and scientific articles referred to herein are incorporated by reference for all purposes.

[0179] Equivalent The present disclosure may be embodied in other specific forms without departing from its spirit and essential characteristics. Therefore, the foregoing embodiments should be considered in all respects as illustrative rather than limiting of the disclosure described herein. The various structural elements of the different embodiments and the steps of the various disclosed methods may be utilized in various combinations and permutations, and all such modifications should be considered as forms of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0180] Sequence Listing SEQ ID NO:1 (AAV9 amino acid sequence) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPD PQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0181] SEQ ID NO:2 (AAV2 ITR) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0182] Array number 3 (SYN1 promoter) AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG

[0183] Array number 4 (CBA promoter) TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTATGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0184] Array number 5 (SV40 polyadenylation signal) GATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAG

[0185] (Consensus Kozak sequence) GCCGCCACC

[0186] SEQ ID NO:7 (CMV enhancer) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG

[0187] SEQ ID NO:8 (SV40 intron) GCTCTAAGGTAAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAT

[0188] SEQ ID NO: 9 (Streptococcus pyogenes IdeS) DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGY RLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0189] SEQ ID NO: 10 (Streptococcus equi IdeZ) MKTIAYPNKPHSLSAGLLTAIAIFSLASSNITYADDYQRNATEAYAKEVPHQITSVWSKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSA RQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0190] SEQ ID NO: 11 (AAV9 nucleic acid sequence)

[0191] SEQ ID NO:12 (CDKL5 brain isoform - isoform 2)

[0192] SEQ ID NO: 13 (CDKL5 standard isoform - isoform 1)

[0193] SEQ ID NO: 14 (CDKL5 Brain Isoform-Isoform 2-Codon Optimized 1)

[0194] SEQ ID NO: 15 (CDKL5 Brain Isoform-Isoform 2-Codon Optimized 2)

[0195] SEQ ID NO: 16 (CDKL5 Brain Isoform-Isoform 2-Codon Optimized 3)

[0196] SEQ ID NO: 17 (CDKL5 standard isoform-isoform 1-codon optimized 1)

[0197] SEQ ID NO: 18 (CDKL5 standard isoform-isoform 1-codon optimized 2)

[0198] SEQ ID NO: 19 (CDKL5 standard isoform-isoform 1-codon optimized 3)

[0199] SEQ ID NO:20 (DTC350)

Claims

1. a) recombinant adeno-associated virus (rAAV) particles; b) buffer; c) a monovalent salt; d) a polyhydric alcohol; and e) triblock copolymer surfactants; 10. A pharmaceutical composition comprising:

2. 10. The pharmaceutical composition of claim 1, having a pH between 7.0 and 7.

4.

3. 2. The pharmaceutical composition of claim 1, wherein the buffer is (i) a phosphate buffer, or (ii) a Tris buffer.

4. The pharmaceutical composition of claim 3, wherein (i) the phosphate is present in the composition at a concentration between 5 mM and 30 mM; or (ii) the Tris is present in the composition at a concentration between 5 mM and 30 mM.

5. The pharmaceutical composition of claim 1 , wherein the polyhydric alcohol is a sugar alcohol.

6. 6. The pharmaceutical composition of claim 5, wherein the sugar alcohol is selected from the group consisting of erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, and xylitol.

7. The pharmaceutical composition of claim 6, wherein the sugar alcohol is sorbitol present in the composition at a concentration between 0.5% and 20%.

8. 10. The pharmaceutical composition of claim 1, wherein the triblock copolymer surfactant is a copolymer of ethylene oxide (EO) and propylene oxide (PO).

9. 9. The pharmaceutical composition of claim 8, wherein the triblock copolymer surfactant is a poloxamer.

10. 10. The pharmaceutical composition of claim 9, wherein the poloxamer is poloxamer 188 (Pluronic® F68).

11. 10. The pharmaceutical composition of claim 9, wherein the poloxamer is present in the composition at a concentration of between 0.0001% and about 0.001%.

12. 10. The pharmaceutical composition of claim 1, wherein the monovalent salt is NaCl, KCl, or a combination thereof.

13. The pharmaceutical composition of claim 12, wherein the composition comprises: (a) NaCl at a concentration between 100 mM and 250 mM; (b) KCl at a concentration between 0.5 mM and 5 mM; or (c) a combination thereof.

14. 14. The pharmaceutical composition of claim 13, further comprising one or more divalent salts.

15. The divalent salt is MgCl ２ , CaCl ２ 15. The pharmaceutical composition of claim 14, wherein the compound is selected from the group consisting of:

16. The pharmaceutical composition of claim 15, wherein the composition comprises: (a) MgCl 2 at a concentration between 0.5 mM and 5 mM; (b) CaCl 2 at a concentration between 0.5 mM and 5 mM; or a combination thereof.

17. rAAV particles were 1 × 10 １０ ~2 x 10 １４ 10. The pharmaceutical composition of claim 1, wherein the composition is present in a concentration of between GC / mL.

18. 2. The pharmaceutical composition of claim 1, wherein the rAAV particles comprise an AAV capsid derived from AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVhu37, or a variant thereof.

19. The buffer solution comprises phosphate at a concentration between 5 mM and 30 mM; The monovalent salt comprises NaCl at a concentration between 100 mM and 250 mM; The polyhydric alcohol comprises sorbitol at a concentration between 1% and 10%; and The triblock copolymer surfactant comprises a poloxamer at a concentration between 0.0001% and 0.001%.

10. The pharmaceutical composition of claim 1, The composition has a pH between 7.0 and 7.

4.

20. The buffer solution comprises Tris at a concentration between 5 mM and 30 mM; The monovalent salt comprises NaCl at a concentration between 100 mM and 250 mM; The monovalent salt comprises KCl at a concentration between 0.5 mM and 5 mM; The polyhydric alcohol comprises sorbitol at a concentration between 1% and 10%; the triblock copolymer surfactant comprises a poloxamer at a concentration between 0.0001% and 0.001%, and 10. The pharmaceutical composition of claim 1, wherein the composition has a pH between 7.0 and 7.

4. wherein said composition further comprises MgCl 2 at a concentration between 0.5 mM and 5 mM and CaCl 2 at a concentration between 0.5 mM and 5 mM.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, for use in intrathecal administration, wherein the intrathecal administration comprises intracisternal, intraventricular, or lumbar administration.

22. The pharmaceutical composition of any one of claims 1 to 20, wherein the rAAV particle comprises an AAV capsid and a vector genome packaged in the AAV capsid, wherein the vector genome comprises a partial or complete coding sequence for CDKL5, or a functional fragment or variant thereof.

23. The pharmaceutical composition described in claim 22, wherein the coding sequence of CDKL5 comprises: (a) a nucleotide sequence selected from SEQ ID NOs: 12 to 19; or (b) a nucleotide sequence that is at least 95% identical to any of SEQ ID NOs: 12 to 19.

24. 23. The pharmaceutical composition of claim 22, wherein the vector genome comprises: (a) the nucleotide sequence of SEQ ID NO:20; or (b) a nucleotide sequence at least 95% identical to SEQ ID NO:

20.

25. A pharmaceutical composition described in any one of claims 1 to 20 for use in a method for treating CDKL5 deficiency disorder (CDD) in a subject, the method comprising administering the pharmaceutical composition to the central nervous system of the subject.

26. 26. The pharmaceutical composition for use of claim 25, wherein said administering comprises administering by intrathecal administration, said intrathecal administration comprising intracisternal, intracerebroventricular, or lumbar administration.

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