Gene Therapy for the Treatment of Cognitive Disorders

Abstract

Methods and compositions for the treatment of cognitive disorders are provided herein.The present disclosure generally relates to the field of methods for delivering gene therapy to the entorhinal / hippocampal region accurately and safely to treat cognitive disorders and other diseases of the central nervous system (CNS).The present disclosure describes the specific parameters for targeting this network of brain regions, including accurate anatomical target, vector concentration and vector volume, and the subject may be at risk of suffering from a condition selected from Alzheimer's disease (AD), mild cognitive impairment, pre-symptomatic AD, frontotemporal dementia, or Lewy body dementia.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 328,934, filed April 8, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Government support statement This invention was made with government support under awards AG043416 and AG010435 from the National Institutes of Health. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Technical Field The present disclosure generally relates to the field of methods for precisely and safely delivering gene therapy to the entorhinal / hippocampal region to treat cognitive disorders and other diseases of the central nervous system (CNS). The present disclosure describes specific parameters for targeting this network of brain regions, including precise anatomical targets, vector concentration and vector volume. Summary of the Invention [Means for solving the problem]

[0004] Summary of disclosure One embodiment of the present disclosure is a method for improving cognitive function in a subject in need thereof, comprising administering a therapeutic peptide or a polynucleotide encoding brain-derived neurotrophic factor (BDNF) to a subject in need thereof at an infusion rate of between about 0.001 ml / min to about 0.015 ml / min and an infusion volume of between about 250 μl to about 750 μl per hemisphere or alternatively both hemispheres, in a dose of about 3×10 11 vg / ml ~ approx. 1×10 13The present invention relates to a method for improving cognitive function in a subject, comprising, alternatively consisting essentially of, or even still consisting of, administering a therapeutic agent to the ventromedial nucleus of a subject at a dose of 100 mg / ml ...

[0005] In some embodiments, the subject is afflicted with a condition selected from Alzheimer's disease (AD), mild cognitive impairment, pre-symptomatic AD, frontotemporal dementia, or dementia with Lewy bodies.

[0006] In one embodiment, the subject being treated is pre-symptomatic and cognitively intact, but at high risk of developing cognitive impairment (e.g., Alzheimer's disease, based on biomarkers such as cerebrospinal fluid testing and brain positron emission tomography imaging). In one embodiment, the method further comprises assaying for these biomarkers from the subject before and / or after administering therapy, and optionally comparing the results to baseline values ​​for the general population or for the subject in particular.

[0007] In some embodiments, the subject is a mammal or a human, monkey, rat, mouse, horse, cat, dog, or sheep.

[0008] In some embodiments, the polynucleotide further comprises an expression vector, and the polynucleotide is administered in the expression vector.Non-limiting examples of such include, for example, plasmid, liposome, lentiviral vector, adenoviral vector, or adeno-associated vector (AAV).Methods for making such vectors are known in the art and are briefly described herein.In some embodiments, the polynucleotide is operably linked to a control nucleotide to drive the expression of the polynucleotide.Non-limiting examples of such control nucleotide include promoter and enhancer element.

[0009] In some embodiments, the administering step comprises, consists essentially of, or even consists of convection-enhanced delivery (CED), in some embodiments, the CED further comprises an infusion catheter having a step distance from the infusion tip of about 0.5 mm to about 2.0 mm and ranges therebetween.

[0010] In some embodiments, the administering step does not substantially deliver to or excludes selected regions of the brain selected from one or more of the following: the presubiculum, the parasubiculum, the subiculum, or the hippocampus. In another aspect, the administering step does not substantially deliver to or excludes selected regions of the brain selected from two or more, three or more, or all of the following: the presubiculum, the parasubiculum, the subiculum, or the hippocampus.

[0011] In some embodiments, the polynucleotide is administered to a subject at three or four injection sites.

[0012] In some embodiments, administration is in one or more doses, each dose containing at least 3×10 11 In some embodiments, administration is in one or more doses, each dose comprising about 3×10 11 vg / ml ~ approx. 5×10 11 vg / ml, approx. 4×10 11vg / ml ~ approx. 6×10 11 vg / ml, approx. 5×10 11 vg / ml ~ approx. 7×10 11 vg / ml, 6 × 10 11 vg / ml ~ approx. 8×10 11 vg / ml, approx. 7×10 11 vg / ml ~ approx. 9×10 11 vg / ml, approx. 8×10 11 vg / ml ~ approx. 1×10 12 vg / ml, approx. 9×10 11 vg / ml ~ approx. 2×10 12 vg / ml, approx. 1×10 12 vg / ml ~ approx. 3×10 12 vg / ml, 2 × 10 12 vg / ml ~ approx. 4×10 12 vg / ml, 3 × 10 12 vg / ml ~ approx. 5×10 12 vg / ml, 4 × 10 12 vg / ml ~ approx. 6×10 12 vg / ml, 5 × 10 12 vg / ml ~ approx. 7×10 12 vg / ml, 6 × 10 12 vg / ml ~ approx. 8×10 12 vg / ml, 7 × 10 12 vg / ml ~ approx. 9×10 12 vg / ml, or 8 × 10 12 vg / ml ~ approx. 1×10 13 In a further aspect, a composition is provided herein comprising a dose of a polynucleotide and / or vector having the aforementioned vg / ml. The composition may further comprise a preservative or cryoprotectant or other agent to facilitate delivery. In a further aspect, the composition is lyophilized.

[0013] One embodiment of the present disclosure is a method for delivering an expression vector to the ventromedial nucleus of a subject in need thereof, comprising: (a) an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip; (b) an injection rate of about 0.001 ml / min to about 0.015 ml / min; (c) an injection volume of about 250 μl to about 750 μl per hemisphere, where the injection is performed at about 3 to about 4 injection sites; and (d) a volume of about 3×10 11 vg / ml ~ approx. 1×10 13 The present invention relates to a method for administering a vector by injection of a vector comprising, consisting essentially of, or even consisting of a dose of 1000 ng / ml of the vector, wherein the delivery avoids the presubiculum, parasubiculum, subiculum, or hippocampal regions, and wherein the subject has a cognitive impairment. In one aspect, the expression vector is delivered using an injection cannula with a step design with a distance of 0.5 mm to 2.0 mm from the injection tip.

[0014] In some embodiments, the expression vector further comprises a therapeutic polynucleotide. In some embodiments, the polynucleotide encodes a protein selected from the group of brain-derived neurotrophic factor (BDNF), palmitoyl-protein thioesterase 1 (PPT1), tripeptidyl peptidase 1, CLN6 (linklin), CLN8, cathepsin D, or MFSD8 or battenin. Other therapeutic proteins with neurological functions are known in the art. In one aspect, the expression vector is delivered using an injection cannula with a step design with a distance of 0.5 mm to 2.0 mm from the injection tip.

[0015] In some embodiments, the expression vector is selected from plasmid, liposome, lentivirus vector, adenovirus vector, or adeno-associated vector (AAV).The method of making such vector is known in the art and is briefly described herein.In some embodiments, the polynucleotide is operably linked to control nucleotide to drive the expression of the polynucleotide.Non-limiting examples of such control nucleotide include promoter and enhancer element.

[0016] In some embodiments, administration is in one or more doses, each dose containing at least 3×10 11 In some embodiments, administration is in one or more doses, each dose comprising about 3×10 11 vg / ml ~ approx. 5×10 11 vg / ml, approx. 4×10 11 vg / ml ~ approx. 6×10 11 vg / ml, approx. 5×10 11 vg / ml ~ approx. 7×10 11 vg / ml, 6 × 10 11 vg / ml ~ approx. 8×10 11 vg / ml, approx. 7×10 11 vg / ml ~ approx. 9×10 11 vg / ml, approx. 8×10 11 vg / ml ~ approx. 1×10 12 vg / ml, approx. 9×10 11 vg / ml ~ approx. 2×10 12 vg / ml, approx. 1×10 12 vg / ml ~ approx. 3×10 12 vg / ml, 2 × 10 12 vg / ml ~ approx. 4×10 12 vg / ml, 3 × 10 12 vg / ml ~ approx. 5×10 12 vg / ml, 4 × 10 12 vg / ml ~ approx. 6×10 12 vg / ml, 5 × 10 12 vg / ml ~ approx. 7×10 12 vg / ml, 6 × 10 12 vg / ml ~ approx. 8×10 12 vg / ml, 7 × 1012 vg / ml ~ approx. 9×10 12 vg / ml, or 8 × 10 12 vg / ml ~ approx. 1×10 13 In one embodiment, the expression vector is delivered using an injection cannula having a step design with a distance of 0.5 mm to 2.0 mm from the injection tip.

[0017] In some embodiments, the subject is a mammal or a human, monkey, rat, mouse, horse, cat, dog, or sheep.

[0018] Methods as disclosed herein can be combined with additional therapeutic and diagnostic methods to further enhance efficacy and reduce toxicity.

[0019] Kits containing compositions, and optionally instructions, for the practice of the methods as described herein are further provided. [Brief description of the drawings]

[0020] [Figure 1] Figure 1: Schematic diagram of the entorhinal cortex and its major afferent and efferent connections with the hippocampus and cerebral cortex. The major projections from the entorhinal cortex (layers 1.1–1.1.1) are to the outer molecular layer of the dentate gyrus (DG) and CM regions of the hippocampus. The CAI region projects back to deeper layers of the entorhinal cortex. The entorhinal cortex also projects directly to cortical areas that are sites of long-term memory storage

[31] . Reprinted from Nagahara et al. (2018) Gene Therapy (2018) 25:104-114.

[0021] [Diagram 2]Figure 2A-2H: Location of the entorhinal cortex (EC) in rhesus monkeys and corresponding MR images. Schematic diagram of the entorhinal cortex across four coronal planes from -2.7 to -14.85 mm relative to bregma (adapted from Paxinos et al.

[34] ) (Figure 2A, Figure 2C-2E). The entorhinal cortex is located on the ventral and medial surfaces of the temporal lobe, with regional landmarks including the rhinorhinal sulcus (RF), perirhinal cortex (PR), amygdala (Am), subiculum region (S), and hippocampus (Hp). T1 and T2 MRI scans (Figure 2B, Figure 2F-2H) with visible landmarks of the rhinorhinal sulcus (RF) shown on T2 images (arrows in Figure 2A, Figure 2B, Figure 2D, Figure 2G). Reprinted from Nagahara et al. (2018) Gene Therapy (2018) 25:104-114.

[0022] [Diagram 3] Figures 3A-3F: Real-time MRI scans of AAV2-BDNF delivery to the entorhinal cortex (entorhinal cortex) of non-human primates (Figures 3A-3C) result in precisely targeted BDNF delivery (Figures 3D-3F). (Figure 3A) An MR-compatible needle is shown passing through the cortex and striatum (arrowheads) to reach the ventral and medial entorhinal cortex (arrows). Gadoteridol spread at the injection site is seen (arrows). The inset shows the gadoteridol signal at higher magnification. (Figure 3B) Corresponding tissue sections from the same animals show BDNF spread by immunolabeling in the same regions predicted by MR imaging. (Figure 3C) Pattern of gadolinium spread within the entorhinal cortex on MR in a different subject, and (Figure 3D) corresponding tissue section. (Figure 3E) Vector spread on MR in a third subject and gadolinium spread along the cortical surface (arrows), and (Figure 3F) corresponding BDNF immunolabeled section. Arrows indicate bands of layer II entorhinal cells containing BDNF. Hp: hippocampus. Scale bars, (Fig. 3D) = 0.5 mm, (Fig. 3E, Fig. 3F) = 1 mm. Reprinted from Nagahara et al. (2018) Gene Therapy (2018) 25:104-114.

[0023] [Figure 4]Figures 4A-4D: AAV2-BDNF and AAV2-GFP transduce primarily neurons. At the injection site, (Figure 4A) BDNF immunolabeling and (Figure 4B) GFP immunolabeling are observed primarily in neurons that are labeled with (Figure 4C) NeuN. (Figure 4D) Overlay. Quantification reveals that 88.2 ± 3.8% of GFP-expressing cells are co-labeled for NeuN. Scale bar = 50 pm. Reprinted from Nagahara et al. (2018) Gene Therapy (2018) 25:104-114.

[0024] [Diagram 5] Figures 5A-5E: Distribution of BDNF-labeled neurons at entorhinal injection sites. (Figure 5A) 13 DNF immunolabeling at AAV2-BDNF injection sites in the entorhinal cortex. (Figure 5B) Map of individual cells immunolabeled for BDNF, and (Figure 5C) Areas of BDNF-containing cells used to quantify the volume of vector distribution. (Figure 5D), (Figure 5E) The volume of injected AAV2-BDNF vector significantly correlates with the volume of tissue containing BDNF-labeled neurons (p<0.001) and the number of BDNF-labeled neurons (p<0.001). Reprinted from Nagahara et al. (2018) Gene Therapy (2018) 25:104-114.

[0025] [Figure 6]Figures 6A-6D: BDNF spreads to the hippocampus. (Figure 6A) BDNF immunoreactivity in the hippocampus of control subjects shows endogenous expression of BDNF in the CA3 stratum lucidum and mossy fiber terminal fields of the hilus region. (Figure 6B) After AAV2-BDNF injection into the entorhinal cortex, BDNF immunoreactivity is seen in the outer molecular layer of the hippocampus (arrowheads). (Figure 6C) Fluorescent labeling shows BDNF immunoreactive fibers in the outer molecular layer (OML), but not in the inner molecular layer (IML) or granule cell layer (GC) of the hippocampus. (Figure 6D) Scatter plots showed that even smaller amounts of AAV2-BDNF injected into the entorhinal cortex could lead to widespread (~70%) increases in BDNF expression in the dentate gyrus. Scale bars: (Figure 6A, Figure 6B) 1 mm; (Figure 6C) 200 μm. Reprinted from Nagahara et al. (2018) Gene Therapy (2018) 25:104-114.

[0026] [Figure 7] Figures 7A-7C: Demonstrating the critical need to target the entorhinal subregion to effectively and safely treat memory disorders. These figures show relatively high vector titers (see Table 2) of 1x1012vg / ml to 1x1013vg / ml from primate study 1004, indicating that this dose is tolerated when targeted precisely to the entorhinal cortex. These injections at titers up to 1013vg / ml resulted in good entorhinal cortex expression without toxicity (seizures) associated with higher doses or vector mistargeting. The arrowheads below indicate the area of ​​BDNF gene expression following vector injection. (Figure 7A) Monkey 21684. (Figure 7B) Monkey 22241. (Figure 7C) Monkey 24647.

[0027] [Figure 8] Figures 8A-8E: In contrast, the figures show that the following injections, limited to the entorhinal cortex only and avoiding surrounding structures, resulted in safe vector administration without seizures: (Figure 8A) Monkey 21011; (Figure 8B) Monkey 25722; (Figure 8C) Monkey 23762; (Figure 8D) Monkey 26169; (Figure 8E) Monkey 22295.

[0028] [Figure 9] Figures 9A-9D: Examples of precise vector targeting in four different monkeys from study 1004; these monkeys did not develop seizures. (Figure 9A) Monkey 21684, 24 months after gene delivery. Arrows indicate the area of ​​entorhinal cortex (EC) transduction. (Figure 9B) Monkey 24647, 24 months after gene delivery. (Figure 9C) Monkey 22241, 24 months after gene delivery. (Figure 9D) Monkey 26169, 2.4 months after gene delivery. Scale bars: A 2 mm, B 1.6 mm, C 1.8 mm, D 2.6 mm.

[0029] [Figure 10] 10A-10B: A new injection cannula design with a step design specifically adapted for the entorhinal cortex to accurately and safely treat memory disorders. The optimal distance is about 0.5 mm to about 2.0 mm from the injection tip. (FIG. 10A) Prior art design. (FIG. 10B) Prior art design. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description of the Disclosure definition The embodiments according to the present disclosure will be described in more detail below. However, the aspects of the present disclosure may be embodied in different forms, and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art. The terms used in the description of this specification are only for the purpose of describing specific embodiments, and are not intended to be limiting.

[0031] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined as such herein. Although not expressly defined below, such terms should be interpreted according to their general meaning.

[0032] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0033] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0034] Unless the context requires otherwise, it is specifically intended that the various features of the present disclosure described herein may be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For example, if a complex is described herein as comprising components A, B, and C, it is specifically intended that any of A, B, or C, or combinations thereof, alone or in any combination, may be omitted and abandoned.

[0035] Unless expressly stated otherwise, all specified embodiments, features, and terms are intended to include both the recited embodiment, feature, or term and their biological equivalents.

[0036] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, or alternatively by a variance of + / -15%, or alternatively by 10%, or alternatively by 5%, or alternatively by 2%, as appropriate. It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalents of such are known in the art.

[0037] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying citations or by Arabic numerals. Full citations for publications identified by Arabic numerals are set forth immediately before the claims. The disclosures of these publications, patents, and published patent specifications are incorporated by reference in their entireties into this disclosure in order to more fully describe the state of the art to which this disclosure pertains.

[0038] The practice of this technique employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0039] As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0040] The term "about" when used herein in reference to a measurable value, such as an amount or concentration, is meant to encompass a range of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% variation of the specified amount.

[0041] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any components, ranges, dosage forms, etc. disclosed herein, are intended to mean that said components, ranges, dosage forms, etc. are suitable for the purpose disclosed.

[0042] Also, as used herein, "and / or" refers to and covers any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0043] The term "adeno-associated virus" or "AAV" as used herein refers to a member of a class of viruses related to this name and belonging to the genus Dependoparvovirus of the family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes are capable of infecting cells from various tissue types. At least eleven consecutively numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the eleven serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV PHP.B. AAV particles comprise, or alternatively consist essentially of, or even consist of three major viral proteins: VP1, VP2, and VP3. In one embodiment, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74.

[0044] "Eukaryotic cells" include, or alternatively consist essentially of, or even consist of, all of the kingdoms of life except the kingdom Monera. They can be easily distinguished through their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotic organisms or organisms whose cells are organized into complex structures by internal membranes and cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise stated, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkeys, cows, pigs, mice, rats, birds, reptiles, and humans, such as HEK293 cells and 293T cells.

[0045] "Prokaryotic cells" usually lack a nucleus or any other membrane-bound organelles and are divided into two regions: bacteria and archaea. Besides chromosomal DNA, these cells can also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of an animal mitochondrion (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus bacteria, E. coli bacteria, and Salmonella bacteria.

[0046] When the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, it should be presumed, without express recitation and unless otherwise intended, that equivalents or biological equivalents of such are intended to be within the scope of the present disclosure. As used herein, the term "biological equivalent thereof" is intended to be synonymous with "equivalent thereof" when referring to a reference protein, antibody, polypeptide or nucleic acid, and is intended to have minimal sequence identity while still maintaining the desired structure or function. Unless specifically recited herein, any polynucleotide, polypeptide or protein referred to herein is also intended to include its equivalent. For example, an equivalent is intended to have at least about 70% homology or identity, or at least 80% homology or identity, and alternatively, or at least about 85%, or alternatively, at least about 90%, or alternatively, at least about 95%, or alternatively, 98% homology or identity over the entire length of the reference sequence, and exhibits substantially the same biological activity as the reference protein, polypeptide or nucleic acid. Alternatively, when referring to a polynucleotide, the equivalent is, in one aspect, a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement, and in a further aspect, a polynucleotide that has the same or a similar degree of activity or function as the reference polynucleotide or its complement.

[0047] A protein or polypeptide equivalent (herein referred to as a reference) has at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) identity to the reference and retains the function and manufacturability of the reference.

[0048] The term "encode," when applied to a nucleic acid sequence, refers to a polynucleotide that is said to "encode" a polypeptide when, in its natural state, or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA of the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and a coding sequence can be deduced therefrom.

[0049] The term "equivalent" or "biological equivalent" is used interchangeably when referring to a particular molecule, biological material or cellular material, and is intended to have minimal homology while still maintaining the desired structure or function.Non-limiting examples of equivalent polypeptides include polypeptides that have at least 60%, alternatively at least 65%, alternatively at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, or alternatively at least 95% identity to a polypeptide or polypeptide sequence, or polypeptides encoded by polynucleotides or their complements that hybridize under high stringency conditions to polynucleotides encoding such polypeptide sequences.High stringency conditions are described herein and are incorporated by reference herein. Alternatively, the equivalent is a polypeptide encoded by a polynucleotide or its complement having at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity, or at least 97% sequence identity to a reference polynucleotide, e.g., a wild-type polynucleotide.

[0050] Non-limiting examples of equivalent polypeptides include polynucleotides having at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 97% identity to a reference polynucleotide. Equivalents also contemplate polynucleotides or their complements that hybridize to a reference polynucleotide under high stringency conditions.

[0051] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. Alignment and percent homology or percent sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST using default parameters. In particular, exemplary programs include BLASTN and BLASTP using the following default parameters: Genetic code=standard; Filter=none; Strand=both; Cutoff=60; Expectation=10; Matrix=BLOSUM62; Description=50 sequences; Filter criteria=high score; Database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity can be determined by incorporating them into clustalW (available at the web address: genome.jp / tools / clustalW / , last accessed January 13, 2017).

[0052] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence has less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0053] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0054] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include, or alternatively consist essentially of, or even consist of, two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can be a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0055] Examples of stringent hybridization conditions include: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times are 5 minutes to 24 hours, with one, two, or more washing steps, with wash incubation times of about 1, 2, or 15 minutes. SSC is a 0.15M NaCl and 15 mM citrate buffer. It will be understood that equivalents of SSC using other buffer systems may be used.

[0056] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells.

[0057] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. "Gene product" or alternatively "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0058] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on being operably linked to elements that contribute to or facilitate the initiation of transcription. "Operably linked" intends that the polynucleotide is positioned in a manner that allows it to function within the cell.

[0059] As used herein, the term "isolated" refers to molecules or biological or cellular material that is substantially free of other substances.

[0060] As used herein, the term "functional" can be used to denote the modification of any molecule, biological material, or cellular material so that it achieves a particular, designated effect.

[0061] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to any length of polymeric form of nucleotide, either ribonucleotide or deoxyribonucleotide.Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or polymer that comprises, or alternatively essentially consists of, or even consists of, purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0062] The term "promoter" as used herein refers to any sequence that regulates the expression of a coding sequence, e.g., a gene. A promoter can be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence that is a region of a polynucleotide sequence where the initiation and rate of transcription are controlled. It can include genetic elements to which regulatory proteins and molecules, e.g., RNA polymerase and other transcription factors, can bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, or the EF1 promoter. In some embodiments, the promoter is a chicken β-actin ("CBA") promoter.

[0063] Further non-limiting exemplary promoters with specific target specificity are provided herein below, including but not limited to CMV, EF1a, SV40, PGK1 (human or mouse), P5, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, and alpha-1-antitrypsin. Synthetically derived promoters may be used for ubiquitous or tissue-specific expression. In addition, promoters from viruses, some of which are mentioned above, such as CMV, HIV, adenovirus, and AAV promoters, may also be useful in the methods disclosed herein. In some embodiments, the promoter is linked to an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include RSV enhancers or CMV enhancers.

[0064] Enhancers are control elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeat of a retrovirus contains both promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that has been placed in close proximity to a gene by genetic engineering (i.e., molecular biology techniques) such that transcription of that gene is directed by the linked enhancer / promoter.

[0065] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains, alternatively consists essentially of, or even consists of an intact replicon, such that the vector can replicate when placed in a cell, for example, by the process of transformation. A vector can be viral or non-viral. Viral vectors include retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papovaviruses, or naturally occurring viruses that have been modified in other ways. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA; DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising, alternatively consisting essentially of, or even consisting of DNA condensed with cationic polymers, such as heterologous polylysine, oligopeptides of defined length, and polyethyleneimine, optionally contained within liposomes; and ternary complexes comprising, alternatively consisting essentially of, or even consisting of viruses and polylysine-DNA.

[0066] "Viral vector" is defined as a recombinantly produced virus or virus particle that contains, alternatively consists essentially of, or even consists of a polynucleotide to be delivered to a host cell, either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0067] "Gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotide into host cell.Examples of gene delivery vehicle include liposome, micelle, biocompatible polymers including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0068] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," "transducing," and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell, or is integrated into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, a number of vectors are known that can mediate the transfer of genes into mammalian cells.

[0069] A "plasmid" is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically confer a selective advantage under a given environmental condition. Plasmids may carry genes that confer resistance to antibiotics that naturally occur in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar circumstances.

[0070] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which also contains a gene that makes the cell resistant to a particular antibiotic, and a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites that allow easy insertion of DNA fragments at this location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers grow bacteria that contain a plasmid carrying the gene of interest. Just as bacteria produce the protein that confers its antibiotic resistance, they can also induce bacteria to produce large amounts of a protein from an inserted gene.

[0071] In embodiments where gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide that includes, alternatively consists essentially of, or even consists of, the viral genome or a portion thereof and the transgene. Adenoviruses (Ad) are a relatively well-characterized homogeneous group of viruses that includes more than 50 serotypes. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the possibility of recombination and generation of wild-type virus. Such vectors are commercially available from sources such as Takara Bio USA (Mountain View, CA), Vector Biolabs (Philadelphia, PA), and Creative Biogene (Shirley, NY). Wild-type AAV has high infectivity and specificity to integrate into the genome of host cells. See Wold and Toth (2013) Curr. Gene. Ther. 13(6):421-433, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470, and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0072] Vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate extra, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcription or translation level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' to the initiation codon to enhance expression.

[0073] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeting viral protein-DNA complexes. Liposomes that also include, alternatively consist essentially of, or even consist of targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be performed by the non-limiting method of protein transfection, and alternatively, culture conditions that can enhance the expression and / or promote the activity of the proteins disclosed herein are other non-limiting methods.

[0074] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminus of newly synthesized secretory or membrane polypeptides or proteins. It acts to target the polypeptide to a specific cellular location, for example, across the cell membrane, into the cell membrane, or into the nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Pat. Nos. 8,853,381, 5,958,736, and 8,795,965.

[0075] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within a viral capsid.

[0076] As used herein, the term "helper" in relation to a virus or plasmid refers to a virus or plasmid that is used to provide additional components required for the replication and packaging of a viral particle or recombinant viral particle, such as the modified AAV disclosed herein. The components encoded by the helper virus can include any gene required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus can encode an enzyme required for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0077] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus.Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells where certain functions are provided by co-infecting helper virus.General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169- 228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Since it is well known that the various serotypes are very closely related, both structurally and functionally, and even at the genetic level, it is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized since the publication date of the reviews (see, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between serotypes along the length of the genome, and heteroduplex analysis revealing the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0078] "AAV vector," as used herein, refers to a vector that comprises, alternatively consists essentially of, or even consists of, one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors, when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products, can be replicated and packaged into infectious viral particles.

[0079] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle that is composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.When the particle comprises, alternatively consists essentially of, or even consists of a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene that is to be delivered to mammalian cells), it is typically referred to as "AAV vector particle" or simply "AAV vector".Therefore, the production of AAV vector particle necessarily includes the production of AAV vector.Because such vector is contained within the AAV vector particle.

[0080] In some embodiments, AAV is a replication-deficient parvovirus, and its single-stranded DNA genome is about 4.7 kb in length, including two 145 nucleotide inverted terminal repeats (ITRs). There are many serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45: 555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively, and the AAV-9 genome is provided in Gao et al., J. Virol., 78: The AAV rh.74 genome is provided in U.S. Pat. No. 6,381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928 (incorporated herein by reference). U.S. Pat. No. 9,434,928 also provides the sequences of capsid proteins and self-complementary genomes. In one embodiment, the genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITR. Three AAV promoters (designated p5, pl9, and p40 from their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and pi 9), combined with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately involved in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0081] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture does not cause cytopathic changes, and natural infection of humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing targeting of many different tissues in vivo. Furthermore, AAV can slowly transduce dividing and non-dividing cells, and persist as transcriptionally active nuclear episomes (extrachromosomal elements) essentially for the life of these cells. The AAV proviral genome is inserted as cloned DNA into a plasmid, which allows the construction of recombinant genomes. Furthermore, signals directing AAV replication and encapsidation of the genome are contained within the ITRs of the AAV genome, such that part or all of the internal ∼4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and hardy virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), so cryopreservation of AAV is less essential. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0082] The recombinant AAV (rAAV) genome of the present disclosure comprises, alternatively consists essentially of, or even consists of, a nucleic acid molecule encoding a therapeutic protein (e.g., BDNF) and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B and AAV rh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also envisioned, such as rAAVs with capsid mutations. See, e.g., Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0083] A "composition" is intended to mean a combination of an active polypeptide, polynucleotide, or antibody with another compound or composition, or active agent (eg, a gene delivery vehicle).

[0084] A "pharmaceutical composition" is intended to include a combination of an active polypeptide, polynucleotide or antibody with an inert or active carrier, such as a solid support, which renders the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0085] As used herein, the term "pharmaceutical acceptable carrier" encompasses standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, as well as any of a variety of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0086] "Subject", "individual" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, and primates, particularly humans. In addition to being useful for treating humans, the present disclosure is also useful for veterinary treatment of companion mammals, exotic animals, and farm animals, including mammals, rodents, and the like. In one embodiment, mammals include horses, dogs, and cats. In another embodiment of the present disclosure, the human is an adolescent or infant under the age of 18.

[0087] "Treating" a disease or "treatment" of a disease includes (1) preventing the disease, i.e., preventing clinical symptoms of the disease from developing in patients who may be predisposed to the disease but who have not yet experienced or exhibited symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or (3) palliating the disease, i.e., causing regression of the disease or its clinical symptoms. In one aspect, the term "treatment" excludes prophylaxis or preventative methods.

[0088] The term "suffering" in conjunction with the term "treatment" refers to a patient or individual who has been diagnosed with or is predisposed to a disease.

[0089] The term "condition" refers to a disorder, illness, disease, or disorder.

[0090] The term "cognitive function" refers to multiple mental abilities including, but not limited to, learning, thinking, reasoning, memory, problem solving, decision-making, and attention.

[0091] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic application, the effective amount will depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to cause partial or complete restoration of the function of a gene that is missing in a subject. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to cause expression of a gene in a subject. Those skilled in the art will be able to determine the appropriate amount depending on these and other factors.

[0092] In some embodiments, effective amount will depend on the size and nature of the application.It will also depend on the nature and sensitivity of the target subject and the method used.Those skilled in the art will be able to determine effective amount based on these and other considerations.Effective amount can include one or more administrations of composition, or alternatively can consist essentially of it, or even consist of it, depending on the embodiment.

[0093] As used herein, the term "administer" or "administration" or "administering" is intended to mean the delivery of a substance to a subject, such as an animal or human. Administration can be in one dose, continuous, or intermittent throughout the course of treatment. Methods for determining the most effective means and dosages of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be performed with the dosage levels and patterns selected by the treating physician, or in the case of pets and animals, the treating veterinarian. Suitable dosage formulations and methods for administering drugs are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health or stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation. In some embodiments, administration is convection-enhanced delivery ("CED").

[0094] As used herein, the term "convection-enhanced delivery" or "CED" refers to a therapeutic strategy to facilitate targeted delivery of pharmaceutical agents to the brain. In some embodiments, CED involves minimally invasive surgical exposure of the brain and placement of an infusion catheter. In some embodiments, the infusion catheter has a step distance of about 0.5 mm to 2.0 mm from the catheter tip. As used herein, the term "step" refers to the extension of the cannula beyond the end of the needle tip.

[0095] As used herein, the term "hemisphere" refers to the two separate cerebral hemispheres of the brain that are connected by the corpus callosum.

[0096] BDNF (brain-derived neurotrophic factor) is a 27 kDa homodimer originally derived from human brain, which promotes the outgrowth of spinal cord sensory neurons. In some embodiments, BDNF supports the survival and outgrowth of sensory neurons, ganglion neurons, dopaminergic neurons, cholinergic neurons, GABAergic neurons, and motor neurons. GenBank M61176 shows the coding sequence (mRNA) of BDNF, last accessed on April 7, 2022.

[0097] MODE FOR CARRYING OUT THE DISCLOSURE

[0098] In a previous study (Nagahara et al. (2018) Gene Therapy 25:104-114), applicants reported that delivery of brain-derived neurotrophic factor (BDNF) gene to the entorhinal cortex is a candidate for the treatment of Alzheimer's disease (AD) to alleviate neurodegeneration associated with memory loss. Precise targeting of the entorhinal cortex in AD is complicated due to the deep location and atrophic state of this brain region. Using an MRI-guided method with convection-enhanced delivery, applicants precisely and reliably targeted AAV2-BDNF delivery to the entorhinal cortex of non-human primates; 86±3% of transduced cells within the targeted region colocalized with the neuronal marker NeuN. The volume of AAV2-BDNF injection (3×10 8 vg / pl) is the number of BDNF-labeled cells and the volume of BDNF immunoreactivity (mm 3 ) BDNF is normally transported from the entorhinal cortex to the hippocampus; Applicants also found that following delivery of a therapeutic BDNF vector to the entorhinal cortex, BDNF immunoreactivity was increased in the hippocampus, achieving distribution of the growth factor through key memory circuits.

[0099] In contrast to this earlier study, the applicant herein describes a specific method for gene therapy to the entorhinal cortex / hippocampus system for the treatment of cognitive impairment or as a prophylactic against impending cognitive impairment. This method identifies a set of new and previously unknown specific parameters required for treating the entorhinal cortex / hippocampus system in humans. These methods are applied to Alzheimer's disease (AD).

[0100] Targeting the entorhinal cortex to treat human cognitive disorders requires gene delivery to the ventromedial nucleus, a subregion of the entorhinal cortex, and avoiding the presubiculum, parasubiculum, subiculum and hippocampus.If these parameters are not followed, treatment will lack efficacy and result in neurotoxicity, including risk of seizures and death.To the applicant's knowledge, the literature has not disclosed the need to target this subregion of the entorhinal cortex, nor the need to avoid vector injection into the presubiculum, parasubiculum, subiculum and hippocampus.

[0101] Vector volumes to target the entorhinal cortex in sufficient volume for effective human treatment may, in one embodiment, range from 250-750 μl per hemisphere divided over three to four injection sites. To the applicant's knowledge, previous literature has recommended a much smaller vector range of only 2.5-25 μl per site, which the applicant now knows to be ineffective. Other published literature has recommended vector doses of 15-130 μl per site (up to 375 μl per hemisphere), which the applicant now knows to be too small a dose and is also erroneous. The applicant's doses are appropriate and, based on the studies described herein, have been determined to be most effective with injections of 30-870 μl per site (360-2610 μl or alternatively 250-750 μl per hemisphere) over three to four sites.

[0102] Applicant also identified new vector concentrations that are effective and safe for use in humans. Applicant determined this to be incorrect and found that 3×10 11Gene therapy vector doses below 1×10 vg / ml are ineffective. 13 To the best of the applicant's knowledge, we have found that doses higher than 1×10 10 vg / ml ~ 1 × 10 15 The literature previously cited vector doses in the range of 3×10 11 Only one dose, 1×10 vg / ml, has been described, and the applicant is now 13 No data from the higher ranges, specified up to 1000 ng / ml, have been described. Thus, specific parameters of vector concentration have not been previously reported, which constitute an important step for successful and safe gene delivery.

[0103] To the applicant's knowledge, the optimal infusion rate parameters have not been identified in the literature. Based on new data from animal subjects, the applicant defines, in one embodiment, an optimal rate of vector infusion of 1-15 μl / min to achieve adequate vector spread and coverage of the intended target. Volumes below this result in ineffective vector distribution in the brain, while volumes above this cause tissue damage and associated toxicity.

[0104] To the applicant's knowledge, the literature has not identified optimal hardware for vector injection to achieve adequate vector spread and coverage of the intended target. Applicant now specifies that the needle required to achieve adequate vector distribution in the target area should have a single widened "step" (widening of outer diameter) located 0.5-2.0 mm from the injection tip to reduce vector reflux and loss up the injection tract.

[0105] In addition to the above, applicants now know that these methods are relevant to treating mild cognitive impairment, a precursor condition of Alzheimer's disease, and to treating patients potentially at risk for developing Alzheimer's disease. Applicants have shown that these methods can be used to treat pre-symptomatic patients who are cognitively intact but at high risk for developing Alzheimer's disease based on biomarkers such as cerebrospinal fluid testing and brain positron emission tomography imaging.

[0106] Treatment

[0107] Applicant provides a method for improving cognitive function in a subject in need thereof, comprising administering a polynucleotide encoding brain-derived neurotrophic factor (BDNF) to a subject in need thereof at an infusion rate of about 0.001 ml / min to about 0.015 ml / min and an infusion volume of about 250 μl to about 750 μl per hemisphere, comprising administering about 3×10 11 vg / ml ~ approx. 1×10 13 In one embodiment, the method includes administering a dose of about 0.5 to about 2.0 mm of an infusion catheter to a subject, the dose being about 0.5 to about 2.0 mm from the infusion tip.

[0108] 1. A method for delivering an expression vector to the ventromedial nucleus of a subject in need thereof, comprising: (a) an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip; (b) an injection rate of about 0.001 ml / min to about 0.015 ml / min; (c) an injection volume of about 250 μl to about 750 μl per hemisphere, the injection being performed at about 3 to about 4 injection sites; and (d) an injection volume of about 3×10 11 vg / ml ~ approx. 1×10 13 Further disclosed herein is a method comprising injecting the vector at a dose of 1000 mg / ml, wherein the delivery avoids the presubiculum, parasubiculum, subiculum, or hippocampal region, and wherein the subject has a cognitive impairment. In a further aspect, the administration further comprises an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip.

[0109] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject has a condition. In some embodiments, the condition is a cognitive disorder. In some embodiments, the cognitive disorder is Alzheimer's disease (AD), mild cognitive impairment, pre-symptomatic AD, frontotemporal dementia, or dementia with Lewy bodies. In some embodiments, the subject is pre-symptomatic and cognitively intact, but at high risk of developing AD based on diagnostic biomarkers, including but not limited to cerebrospinal fluid testing and brain positron emission tomography imaging.

[0110] The titer of the expression vector administered in the methods of the present disclosure will vary depending, for example, on the particular vector, the mode of administration, the goal of treatment, the individual, and the cell type being targeted, and can be determined by standard methods in the art. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered at least about 3×10 11 vg / ml, approx. 4×10 11 vg / ml, approx. 5×10 11 vg / ml, approx. 6×10 11 vg / ml, approx. 7×10 11 vg / ml, approx. 8×10 11 vg / ml, approx. 9×10 11 vg / ml, approx. 1×10 12 vg / ml, approx. 2×10 12 vg / ml, approx. 3×10 12 vg / ml, approx. 4×10 12 vg / ml, approx. 5×10 12 vg / ml, approx. 6×10 12 vg / ml, approx. 7×10 12 vg / ml, approx. 8×10 12 vg / ml, approx. 9×10 12 vg / ml to approximately 1 × 10 13 In some embodiments, the dose of the expression vector is administered in a range of up to about 5×10 11 vg / ml, approx. 4×1011 vg / ml ~ approx. 6×10 11 vg / ml, approx. 5×10 11 vg / ml ~ approx. 7×10 11 vg / ml, 6 × 10 11 vg / ml ~ approx. 8×10 11 vg / ml, approx. 7×10 11 vg / ml ~ approx. 9×10 11 vg / ml, approx. 8×10 11 vg / ml ~ approx. 1×10 12 vg / ml, approx. 9×10 11 vg / ml ~ approx. 2×10 12 vg / ml, approx. 1×10 12 vg / ml ~ approx. 3×10 12 vg / ml, 2 × 10 12 vg / ml ~ approx. 4×10 12 vg / ml, 3 × 10 12 vg / ml ~ approx. 5×10 12 vg / ml, 4 × 10 12 vg / ml ~ approx. 6×10 12 vg / ml, 5 × 10 12 vg / ml ~ approx. 7×10 12 vg / ml, 6 × 10 12 vg / ml ~ approx. 8×10 12 vg / ml, 7 × 10 12 vg / ml ~ approx. 9×10 12 vg / ml, or 8 × 10 12 vg / ml ~ approx. 1×10 13 In some embodiments, the dose is at least 3×10 11 In a further embodiment, the administration further comprises an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip.

[0111] In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered at an infusion rate of about 0.001 ml / min to about 0.015 ml / min. The infusion rate can range from at least about 0.001 ml / min, about 0.0015 ml / min, about 0.002 ml / min, about 0.0025 ml / min, 0.003 ml / min, about 0.0035 ml / min, 0.004 ml / min, about 0.0045 ml / min, 0.005 ml / min, about 0.0055 ml / min, 0.006 ml / min, about 0.0065 ml / min, 0.007 ml / min, about 0.0075 ml / min, 0.008 ml / min, about 0.0085 ml / min, 0.009 ml / min, about 0.0095 ml / min, about 0.01 ml / min, to about 0.015 ml / min. In a further aspect, the administration further comprises an infusion catheter having a step distance of about 0.5 mm to about 2.0 mm from the infusion tip.

[0112] In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered at an injection volume of about 250 μl to about 750 μl per brain hemisphere. The injection volume can range from at least about 250 μl, about 275 μl, about 300 μl, about 325 μl, about 350 μl, about 375 μl, about 400 μl, about 425 μl, about 450 μl, about 475 μl, about 500 μl, about 525 μl, about 550 μl, about 575 μl, about 600 μl, about 625 μl, about 650 μl, about 675 μl, about 700 μl, about 725 μl, to about 750 μl. In some embodiments, the injection volume is administered at about three to about four injection sites. In a further aspect, the administration further comprises an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip.

[0113] In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times per week. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 times per month. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject at least every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks. In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered to a subject for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months.In a further aspect, the administration further comprises an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip.

[0114] In some embodiments, the method disclosed herein comprises administering locally any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein. In some embodiments, the method disclosed herein comprises administering any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein to one or more tissues. In some embodiments, the tissue is selected from muscle tissue, epithelial tissue, connective tissue, and nervous tissue. In some embodiments, the tissue is the brain. In some embodiments, the tissue is a subregion of the entorhinal cortex of the brain. In some embodiments, the subregion is the ventromedial nucleus. In some embodiments, the administration avoids the presubiculum, parasubiculum, subiculum, and hippocampus of the brain.

[0115] In some embodiments, the methods disclosed herein include administering any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein by convection-enhanced delivery (CED). In some embodiments, the CED includes an infusion catheter having a step distance of about 0.5 mm to about 2.0 mm from the infusion tip. In some embodiments, the step distance can range from at least about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1.0 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm to about 2.0 mm. In some embodiments, the step reduces reflux and loss up the infusion tract.

[0116] In some embodiments, the polynucleotide encodes brain-derived neurotrophic factor (BDNF). In some embodiments, the polynucleotide further comprises an expression vector. In some embodiments, the polynucleotide is operably linked to a control nucleotide. In some embodiments, the expression vector is a lentiviral vector, an adenoviral vector, or an adeno-associated vector (AAV).

[0117] In a further aspect, the administration further comprises an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip.

[0118] Applicants demonstrate that these methods may be used to treat pre-symptomatic patients who are cognitively intact but who are at high risk of developing Alzheimer's disease based on biomarkers such as cerebrospinal fluid testing and brain positron emission tomography imaging.

[0119] As will be apparent to one of skill in the art, the methods and compositions described above can be combined with other therapeutic compositions and agents for the treatment of the disclosed diseases or conditions.

[0120] Methods for producing AAV vectors

[0121] The method of making adeno-associated virus (AAV) vector (e.g., virus or virus particle) is disclosed herein, and the supplementary method of making AAV vector is known in the art.For example, such method is disclosed in, for example, WO 01 / 83692, which is incorporated herein by reference in its entirety.The general principle of AAV production is outlined in, for example, Carter, Current Opinions in Biotechnology 1533-1539, 1992; and Muzyczka, Curr. Topics in Microbial. and Immunol. 158:97-129, 1992, each of which is incorporated herein by reference in its entirety.Various approaches to producing AAV have been described in Ratschin et al., Mol. Cell. Biol. 4:2072, 1984; Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466, 1984; Tratschin et al., Mol. Cell. Biol. 5:3251, 1985; McLaughlin et al., J. Virol., 62:1963, 1988; and Lebkowski et al., Mol. Cell. Biol., 7:349, 1988; Samulski et al., J. Virol., 63:3822-3828, 1989; U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO 95 / 13392;WO 96 / 17947;PCT / US98 / 18600;WO 97 / 09441(PCT / US96 / 14423);WO 97 / 08298(PCT / US96 / 13872);WO 97 / 21825(PCT / US96 / 20777);WO 97 / 06243(PCT / FR96 / 01064);WO 99 / 11764;Perrin et al., Vaccine 13:1244-1250, 1995;Paul et al., Human Gene Therapy 4:609-615, 1993;Clark et al., Gene Therapy 3:1124-1132, 1996; U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595, each of which is incorporated herein by reference in its entirety. In some embodiments, a method for producing an adeno-associated virus (AAV) vector comprises transducing a cell with any of the AAV packaging systems disclosed herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some aspects, the cell is a recombinant cell that stably expresses the adeno-associated virus rep and cap genes. In some embodiments, the method further comprises culturing the cell to generate a population of transduced cells.In some embodiments, the method further comprises collecting a supernatant from the population of transduced cells. In some embodiments, the method further comprises subjecting the supernatant to one or more purification steps to produce a purified AAV vector sample, the AAV vector sample being substantially free of cellular debris and proteins. Alternatively or additionally, the method further comprises lysing the population of transduced cells to produce a cell lysate. In some embodiments, the method further comprises subjecting the cell lysate to one or more purification steps to produce a purified AAV vector sample, the AAV vector sample being substantially free of cellular debris and proteins. In some embodiments, the purity of the purified AAV vector sample is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0122] Viruses, such as AAV, can be packaged using viral packaging systems, such as retrovirus, adenovirus, herpesvirus, or baculovirus packaging systems.In some embodiments, packaging is achieved by using helper virus or helper plasmid and cell line.Helper virus or helper plasmid comprises elements and sequences that facilitate the delivery of genetic material to cells.In another aspect, helper plasmid or the polynucleotide that comprises, alternatively consists essentially of, or even consists of helper plasmid is stably integrated into the genome of packaging cell line, so that packaging cell line does not need further transfection with helper plasmid. EXAMPLES

[0123] Vector creation and preparation

[0124] Prior art methods can be used for vector production. The AAV2-BDNF vector was produced by UNC Vector Core (University of North Carolina) as disclosed in Nagahara et al. (2018) and reproduced herein. The vector genome consists of human BDNF cDNA with a human cytomegalovirus (CMV) enhancer, chicken β-actin promoter and CAG promoter consisting of a splice donor, an intron, and a rabbit β-globin splice acceptor. The vector is designed similarly to the AAV2 vector used in phase 1 and phase 2 gene therapy trials [Tuszynski et al., JAMA Neurol. 2015;Arvanitakis et al., AbstrAm Acad Neurol. 2007;Rafii et al., Atzheimers Demerit. 2014.]. Briefly, vector particles were prepared by transient transfection of plasmid DNA into 293 cells and purified by CsCl2 centrifugation, FPLC, and sterile filtration. AAV2-BDNF vector was injected at 3 × 10 12 For injections, AAV2-BDNF was delivered at a titer of 3 × 10 vg / ml and aliquoted into 100 μl volumes. 11 The MR contrast agent gadoteridol (Prohance, Bracco Diagnostic, Princeton, NJ) was mixed with the vector at a final concentration of 1 mM. For a subset of subjects (N=9), 0.03 × 10 12 AAV2-GFP was also administered at a titer of 10000 vg / ml to confirm the spread of the viral vector upon cell transduction.

[0125] surgical exposure

[0126] The prior art method with the modifications disclosed by the applicant can be used for the surgical method. As disclosed in Nagahara et al. (2018) and reprinted herein, the animal's head was placed in a stereotaxic frame after induction of anesthesia and flexed in a prone position. The head was shaved and cleaned using Nolvasan solution and alcohol. A sterile field was created and a midline incision was made through the skin, muscle, and fascia by electrocautery (Surgistat Electrosurgery, Valleylab Inc., Boulder, CO). Gentle retraction of the fascia and muscle allowed the skull to be exposed above the cortical entry sites. Small burr holes were drilled according to the stereotaxic coordinates to expose the dura above each of the intended injection sites. The infusion system included (ii) a CO2 “ball-joint” type array (Hayes Manufacturing Services Inc., USA) fixed to the skull by titanium screws over the craniotomy, and (iii) a custom-designed cannula (Richardson et al. [Richardson et al., Mol Ther. 2011; Richardson et al., Stereotact Fund Neurosurg. 39. 2011.]). The custom-designed ceramic fused silica anti-reflux cannula

[44] consists of an 8-25 cm ceramic section (1.68 mm outer diameter (OD)) in the main part of the shaft, and an 18 mm fused silica section tapering to 0.7 mm OD. The final 3 mm section is a fine fused silica lip (0.36 mm OD). Briefly, the cannula is connected to a loading line containing the infusate, and the flow is regulated with a 1 ml syringe attached to an MRI-compatible infusion pump. A target is selected and an optimal trajectory is established using neuronavigation software on the animal's baseline MRI image. The distance from the target to the top of the guide stem is then determined in silica and a depth stop is fixed at the insertion distance within the cannula. After determining the optimal injection parameters, the cannula is manually inserted through the guide stem of the array into the target.

[0127] MRI-guided injection procedure

[0128] In a prior art method (Nagahara et al. (2018)) that can be modified as disclosed herein, the applicant used a 3 Tesla Siemens Magnetom Avanto scanner (Siemens Medical Solutions, Erlangen, Germany) equipped with Siemens resident software. The vector injection procedure was performed using three types of scanning protocols that were varied throughout the gene delivery procedure: a T1 protocol to optimize visualization of brain structures for the purpose of needle targeting, and a modified T2 protocol to obtain rapid scans to assess vector spread, as well as an MP-RAGE protocol to optimize visualization of white matter structures and vector spread (repetition time: 2110 ms; echo time: 3.6 ms; flip angle: 15°; number of excitations: 1 (3 repetitions); matrix: 240×240; field of view: 240×240×240).

[0129] The animals were sedated by intramuscular injection of ketamine (10 mg / kg IM) and medetomidine (0.015 mg / kg IM), intubated, and an intravenous line was established with a 22-24 gauge catheter placed in the cephalic or saphenous vein to deliver isotonic fluids at a rate of 5–10 ml / kg / h. A stable level of anesthesia was maintained by delivering isoflurane inhalation anesthesia (Aerrane, Omeda PPD Inc., Liberty, NI) at 1–3%. Subsequently, after being placed in an MRI-compatible stereotactic frame in a supine position, two burr holes were placed for the implantation of a ball-joint array consisting of a base (attached to the skull) and a guide tube array (a cylinder with three holes). [Salegio et al., Methods Mol Biol. 2016.]. The monkeys were then transferred to the MRI scanner room. The infusion needle consisted of a ceramic silica anti-reflux cannula with a 1 mm step at the distal end to prevent vector reflux onto the cannula (MRI Intervention, Irvine). [San Sebastian et al., Mol Ther Methods Clin Dev. 2014.]. The infusion needle was then connected to a 1 ml syringe pump (Medfusion 3500 Syringe Pump, Medfusion, St Paul, MN) using 36 inches of high pressure intravenous tubing. The vector was diluted to 3.0 × 10 11 In nine subjects, one-tenth of the vector injection volume was adjusted to a final concentration of AAV2-GFP (3 × 10 11 vg / ml), and 90% of the vector was AAV2-B DNF (3 × 10 11vg / ml); these monkeys allowed for the analysis of the types of cells in the brain that were transduced by the virus. In the first subject, the injection needle was placed in a skull-based stereotactic frame and advanced in the brain to a point calculated to be located in the mid-striatum; the accuracy of the needle trajectory and depth was then confirmed with a T2 scan. As none of these intermediate trajectory scans required correction of the needle trajectory, in all subsequent subjects the injection needle was lowered into the entorhinal cortex itself, stopping at a point calculated in the first MRI scan to be located 1 mm dorsal to the intended target region. A weighted set of T1, MP-RAGE and T2 images was then obtained. Based on these images, the applicant then advanced the injection needle to the distance required to reach a point in the entorhinal cortex located 1 mm from the ventral brain surface (Figure 2).

[0130] Monkeys received one to three injection sites in the entorhinal cortex per side of the brain. For one injection per side, the injection was placed at approximately the midpoint of the anterior-posterior length of the hippocampus. For entorhinal cortices that received two to three injections, an attempt was made to spread the vector to the majority of the entorhinal cortex volume. For this study, 34 entorhinal cortices were injected with AAV2-BDNF (3 × 10 11 vg / mL): 18 received a single injection site per side of the brain, 9 received two injections, and 7 received three injections.

[0131] At the point when the infusion needle was about to penetrate the brain, the infusion pump was turned on at a rate of 3 μU / min to maintain positive pressure to prevent needle blockage as it advanced through the brain to the target. Once the final target was confirmed by MRI, the infusion rate was adjusted with a gradient of 1 to a maximum of 3 pl / min to achieve vector spread through the entorhinal cortex region located in a 1 mm thick MRI slice that included the infusion needle. Rather than injecting a preset volume of vector, applicants continued the infusion until the intended target region (approximately 5 mm of the rostral-caudal extent of the entorhinal cortex) was covered by gadoteridol. Scans were acquired continuously as previously described [Su et al., Mot Ther. 2010; San Sebastian et al. Mol Ther Methods Clin Dev. 2014.]. The range of vector volumes injected per site was 15 to 160111. Vital signs were continuously monitored throughout the procedure.

[0132] After completion of the injection, the subjects were returned to the operating room for removal of the skull-based stereotactic frame. The animals were subsequently given intramuscular injections of NSAID (meloxicam) and buprenorphine (Buprenex) the day after the convection-enhanced delivery (CED) injection as part of post-procedural analgesia management. Once the animals were returned to their cages, they were evaluated twice daily for 5 days by veterinary staff. Detailed standardized forms were completed for each animal, including assessments of surgical site integrity, edema, infection, balance, locomotion, posture, food intake, and fecal and urinary output. No abnormalities or signs of discomfort were reported.

[0133] Treatment of cognitive impairment

[0134] The applicant's work reported herein has resulted in substantial modifications to methods previously used for gene delivery to the entorhinal cortex / hippocampus circuit to treat cognitive disorders. These modifications are based on expanding clinical experience in 1) primate studies delivering gene therapy vectors to the entorhinal cortex and hippocampus, and 2) three current gene therapy studies using MR guidance, convection-enhanced delivery (CED) and gadoteridol co-infusion. These considerations take into account the new findings that the relationship between infusate volume and vector distribution depends on the precise needle location in the entorhinal cortex, variable vector spread in gray vs. white matter, the presence of perivascular spaces (which act as vector sinks during vector infusion), the irregular anatomy of the entorhinal cortex, and the possibility of vector reflux along the infusion tract. These factors require a clearly wider range of vector volumes, a specific rate of vector infusion, and a specific vector dose (concentration) to effectively fill the target brain structures.

[0135] The volume of the human entorhinal cortex is estimated to be 1500 mm by both high-resolution brain MR imaging and anatomical measurements. 3[Hasan et al., J. Neuroimaging 26 (2015);Fischl et al., Neuroimage 47, (2009);Juottonen et al., Neurobiol. Aging 19, (1998);Bunce et al., J Alzheim Dis 30, (2012)]. The ratio of vector infusion volume to volume of distribution in the brain (Vi / Vd) ranges from 1:1 to 1:3 in the gray matter (a ratio of 1:1 means that the volume of infusion and the volume of distribution are the same, and a ratio of 1:3 means that the volume of infusion covers three times the volume of brain tissue). Thus, without being bound by theory, applicants calculate that a total vector volume of 500-1500 μl is required to fill the entorhinal cortex. Applicants required an infusion of 250-750 μl per brain hemisphere, aiming to fill at least half of this volume. Applicants divided this vector volume among three to four injection sites, limiting the maximum injection volume per hemisphere to 750 μl. As with other intracranial gene therapy trials in the brain, adequate coverage requires a range of injection volumes per site to most effectively fill the irregular structures of the entorhinal cortex. MRI imaging can be used to track vector distribution and assess vector distribution in real time to guide injection volumes within the described ranges.

[0136] In monkeys, the applicant administered 3×10 11 The vector was injected at a concentration of 1000 µg / ml to achieve 50–100% filling of the entorhinal cortex. 3 ) [5] is one-sixth the volume of the human entorhinal cortex. Projecting our preclinical injection volumes from monkey to human to achieve 50% coverage of EC, the corresponding injection volumes in humans range from 360-2610 μl or alternatively 250-750 μl per hemisphere.

[0137] [Table 1]

[0138] The volumes originally quoted by Applicant for human injections (maximum 125 μl per site versus maximum 375 μl per hemisphere) were too conservative. Applicant's detailed review of new results from additional monkeys and recent expanded experience in vector delivery to humans has led to these new guidelines: larger volumes than originally quoted are required to achieve sufficient filling of target structures in humans.

[0139] The revised parameters are based on testing in an additional 25 monkeys that allowed the applicant to generate this particular expertise. Previous doses described in the literature were found to be ineffective (1 x 10e1), and 3 x 10 11 It has been found that a minimum dose of 10 ...

[0140] Up to four injections per hemisphere are required to effectively treat the human brain. Appropriate administration volumes in humans include the range of 360-2610 μl per hemisphere. The literature describes lower vector doses (1×10e10-1×10e11) that applicant now knows to be ineffective, and higher doses (greater than 3×10e12) that applicant now knows to be too high and would cause toxicity. The literature also describes methods requiring injections in the range of 15-125 or 15-130 μl / site, which means that the maximum volume injected at each site is 125 μl, for a maximum total volume per hemisphere of 375 μl.

[0141] The extended survival in monkeys of up to 24-30 months identified issues of mistargeting and seizures using previously published methods. To the applicant's knowledge, this is essential information that has not been published regarding achieving safety of vector delivery. To safely deliver vector, it is essential that delivery is achieved within the EC region and limited to its most ventromedial part, excluding any vector injection into the subiculum of the entorhinal cortex and parasubiculum of the entorhinal cortex, and also excluding direct injection into the hippocampus. Animals receiving injections that do not fall within these parameters developed seizures.

[0142] Based on these unpublished long-term monkey studies, applicants also believe that 11 ~1×10 13 A vector dose range of 10000 vg / ml was identified as the maximum dose range that was safe and tolerated.

[0143] Nagahara et al. (2018) Gene Therapy (2018) 25:104-114 teach the use of MRI guidance with gadoteridol infusion to reliably inject AAV2-BDNF into this small brain region. To the applicant's knowledge, the prior art does not provide specific methods for targeting the lower parts of the entorhinal cortex (ventromedial region) to avoid the presubiculum, parasubiculum, subiculum and hippocampus, and the literature does not address proper volume considerations and vector concentrations. For example, the literature does not disclose specific details regarding: 1) effective vector concentrations for use in humans (e.g., to the applicant's knowledge, 3×10 11 Although there are published reports of 100% EDTA of approximately 3×10 vg / ml, the present applicant has determined that 11 vg / ml, 1×10 12 vg / ml up to 1×10 13 vg / ml human dose is recommended; to Applicants' knowledge, the higher doses that Applicants have tested in monkeys have not been reported or published; 2) the effective volume for injection into humans.

[0144] To the applicant's knowledge, the literature has not identified optimal infusion rate parameters to achieve vector spread within the target region of the brain. Vector infusion rates using a continuous infusion pump have been determined to fall within a specific range of 1-15 μl / min in non-human primates. This infusion rate achieves adequate vector spread and coverage of the intended target. Lower rates do not spread far enough within the target structures, and higher rates result in leakage from the intended target region, posing a safety risk.

[0145] The choice of injection needle design for gene delivery to the brain should depend on testing of injection needles specifically designed for various regions of the brain. In the entorhinal cortex, applicants have found that a needle "step" design, in which the first few millimeters of the needle are thin followed by an increase in needle diameter, is an effective means of preventing backflow of injection up the needle insertion tract. In the entorhinal cortex region, the optimal needle design includes a single widened "step" (enlarged outer diameter) located at a distance of 1-5 mm from the tip of the injection needle. This reduces backflow and loss of vector up the injection tract. Unintended spread can compromise safety. This knowledge is essential for the effective implementation of gene therapy vectors into this brain region.

[0146] Vector concentration: lower doses are ineffective, higher doses are toxic: ideal ranges are established

[0147] Minimum effective vector titer is 3 x 10 11 The choice of vg / ml is 1×10 11 This is based on a previous primate study 1006 (see Table 2) which found that a concentration of 1 × 10 vg / ml was insufficient to effectively treat the target brain region, the entorhinal cortex. The human entorhinal cortex is 30 mm in length from rostral to caudal, but 11 Vector injected at titers of 1000 vg / ml resulted in BDNF expression only within 1 mm of the injection site. 11This was also the case when injection volumes comparable to those used in monkeys receiving higher vector titers of 1×10 11 We conclude that the vg / ml potency is insufficient to treat the full volume of degenerating human entorhinal cortex in AD.

[0148] In primate study 1006 (see Table 2), Applicant has 11 The vector titer in vg / ml was also tested. The vector injected at this concentration was 1×10 11 Injection volumes comparable to 1000 mg / mL resulted in BDNF expression up to 8 mm from the injection site (mean spread per site was 5.21 ± 0.71 mm). Primate study 1006 (see Table 2) also used 3 × 10 11 A vector concentration of 10 ...

[0149] 1×10 12 vg / ml ~ 1 × 10 13 Regarding higher vector titers of 10 vg / ml: primate study 1004 (see Table 2) showed that this dose was tolerated when precisely targeted to the entorhinal cortex. 13 These injections at titers up to 5000 ng / ml resulted in good entorhinal expression without toxicity (seizures) associated with higher doses or vector mistargeting. The arrowheads below indicate the area of ​​BDNF gene expression following vector injection. See Figure 7.

[0150] This study demonstrates that infusion of AAV2-BDNF into regions adjacent to the entorhinal cortex (hippocampus, amygdala, presubiculum, parasubiculum, and subiculum) in five monkeys resulted in a safety issue (seizures). This toxicity was previously unknown and unexpected, and underscores the need for precise injection into targeted regions of the entorhinal cortex to safely and effectively treat memory disorders.

[0151] A new range of vector volumes is needed to accurately and safely treat memory disorders

[0152] The volume of the human entorhinal cortex is estimated to be 1500 mm by both high-resolution brain MR imaging and anatomical measurements. 3[Hasan et al., J. Neuroimaging 26 (2015);Fischl et al., Neuroimage 47, (2009);Juottonen et al., Neurobiol. Aging 19, (1998);Bunce et al., J Alzheim Dis 30, (2012)]. The ratio of vector infusion volume to volume of distribution in the brain (Vi / Vd) ranges from 1:1 to 1:3 in the gray matter (a ratio of 1:1 means that the volume of infusion and the volume of distribution are the same, and a ratio of 1:3 means that the volume of infusion covers three times the volume of brain tissue). Thus, applicants calculate that a total vector volume of 500-1500 μl is required to fill the entorhinal cortex. Applicants' aim in this study is to fill at least half of this volume, which would require an infusion of 250-750 μl per brain hemisphere. Applicants propose to split this vector volume among three to four injection sites and propose to limit the maximum injection volume per hemisphere to 750 μl. As with other intracranial gene therapy trials in the brain, adequate coverage requires a range of injection volumes per site to most effectively fill the irregular structures of the entorhinal cortex. Furthermore, because Applicants use MR imaging to track vector distribution, Applicants believe they can accurately assess vector distribution in real time to guide injection volumes within the described ranges.

[0153] In monkeys, applicants have used 3 × 10 11 The vector was injected in a volume of 60–435 μl at a vector concentration of 1000 μg / ml. 3) [Paxinos et al., The Rhesus Monkey Brain in Stereotaxic Coordinates. (Academic Press, 1989)] is 1 / 6 the volume of the human entorhinal cortex. Predicting our preclinical injection volumes from monkey to human to achieve 50% coverage of EC, the corresponding injection volumes in humans range from 250 to 2610 μl per hemisphere. These volumes are much higher than originally anticipated and disclosed in the literature, and new parameters were established by detailed monkey empirical testing and by our recent human experience. Considering the monkey to human conversion factor has been important in our clinical experience to date, where higher volumes than originally predicted were required to achieve adequate filling of target structures in humans.

[0154] To treat memory disorders accurately and safely, the infusion rate must be precisely controlled. Previous methods of delivering AAV intraparenchyma by simple injection were ineffective in distributing the vector to significant volumes of target tissue. In contrast, a technique known as CED (convection-enhanced delivery) uses specially designed anti-reflux cannulas to allow high injection pressures to be applied to the cannula tip, thereby overcoming interstitial pressures within the brain and allowing the infusate to penetrate many volumes of the target area in a uniform manner. In addition, a chelated gadolinium contrast agent (gadoteridol) is included in the infusate to monitor the distribution of the infusate by magnetic resonance (MR) imaging. The distribution of gadoteridol closely matches that of the injected AAV2 vector, and no evidence of toxicity has been seen. 7~9 Preclinical and clinical studies have shown that simultaneous delivery of gadoteridol, a widely used contrast agent for MR imaging that is commercially available for intravenous administration and used off-label in this protocol, and real-time MR imaging helps ensure exposure of the target area to the test agent while minimizing exposure of non-target CNS tissues. 10、11MR images acquired during the CED procedure are analyzed postoperatively to measure the volume of gadoteridol contrast enhancement. The total volume of gadoteridol distribution and the percentage of entorhinal cortex coverage by gadoteridol are calculated. Correlations between coverage and other outcome measures, such as clinical cognitive measures, can be assessed.

[0155] In extensive preclinical studies in monkeys using infusion parameters ranging from 0.1 μl / min to 15 μl / min, applicants have identified a new range of infusion rates: 1-15 μl / min that supports consistent, safe and effective gene delivery to the entorhinal cortex to treat memory disorders. Lower infusion rates result in insufficient spread according to MRI, while higher infusion rates cause localized tissue damage. To the best of applicants' knowledge, this range is novel and inventive over the prior art, and this rate is critical to the implementation of this technology. See Table 2.

[0156] [Table 2-1] [Table 2-2] [Table 2-3]

[0157] To accurately and safely treat memory disorders, an infusion cannula with a step design specifically adapted to the entorhinal cortex is needed.

[0158] As mentioned above, CED (convection-enhanced delivery) uses a specially designed anti-reflux cannula to allow high injection pressure to be applied to the cannula tip, thereby overcoming interstitial pressure in the brain and allowing the infusate to penetrate many volumes of the target area in a uniform manner. The step design in the infusion catheter has been developed taking into account the anatomy of the gray and white matter of the entorhinal cortex target area in particular. By testing various step designs, the applicant found that the optimal step distance is between 0.5 and 2.0 mm from the infusion tip. This distance is essential to prevent backflow into the subcortical white matter and loss of vector in the wrong target. This step distance was not obvious and was the result of empirical testing in the entorhinal cortex of the rhesus monkey brain. As shown below, previous step designs had longer distances to targets such as the striatum in the treatment of Parkinson's disease. This design is considered ineffective when applied to the entorhinal cortex.

[0159] Equivalent

[0160] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0161] The present invention illustratively described herein may be suitably practiced in the absence of any element or elements, or any limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing" and the like are to be read broadly and without limitation. In addition, the terms and expressions used herein are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention as claimed.

[0162] Accordingly, it should be understood that the materials, methods, and examples provided herein are representative of preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention.

[0163] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a condition or negative limitation that removes any subject matter from the genus, regardless of whether the excluded material is specifically recited herein.

[0164] Additionally, when features or aspects of the invention are described in terms of a Markush group, one of skill in the art will recognize that the invention is also described in terms of any individual members or subgroups of members of the Markush group.

[0165] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0166] Other embodiments are within the scope of the following claims. References: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

Claims

1. A composition for use in a method for improving cognitive function in a subject requiring such improvement, comprising a polynucleotide encoding brain-derived neurotrophic factor (BDNF), wherein the method involves administering the polynucleotide encoding brain-derived neurotrophic factor (BDNF) at an infusion rate of about 0.001 ml / min to about 0.015 ml / min and an infusion volume of about 250 μl to about 750 μl per hemisphere, for a total of about 3 × 10 １１ vg / ml ~ approx. 1×10 １３ A composition characterized by comprising the step of administering the dose to the ventromedial nucleus of the subject at a dose of vg / ml.

2. The composition according to claim 1, wherein the subject is suffering from a condition selected from Alzheimer's disease (AD), mild cognitive impairment, pre-symptomatic AD, frontotemporal dementia, or Lewy body dementia.

3. The composition according to claim 1 or 2, wherein the subject is a mammal or a human.

4. The composition according to claim 1 or 2, wherein the polynucleotide further comprises an expression vector, and the polynucleotide is operably linked to a control nucleotide for driving the expression of the polynucleotide.

5. The composition according to claim 1 or 2, wherein the administration step is performed by convection-enhanced delivery (CED).

6. The composition according to claim 1 or 2, wherein the CED includes an injection catheter having a step distance of about 0.5 mm to about 2.0 mm from the injection tip.

7. The composition according to claim 1 or 2, wherein the administration step is not directed to the anterior hippocampal stent, parahippocampal stent, hippocampal stent, or hippocampus.

8. The composition according to claim 1 or 2, characterized in that polynucleotides are administered to three or four injection sites.

9. The aforementioned dose is at least 3 × 10 １１ The composition according to claim 1 or 2, comprising vg / ml.

10. The administration is about 3×10 １１ vg / ml to about 5×10 １１ vg / ml, about 4×10 １１ vg / ml to about 6×10 １１ vg / ml, about 5×10 １１ vg / ml to about 7×10 １１ vg / ml, 6×10 １１ vg / ml to about 8×10 １１ vg / ml, about 7×10 １１ vg / ml to about 9×10 １１ vg / ml, about 8×10 １１ vg / ml to about 1×10 １２ vg / ml, about 9×10 １１ vg / ml to about 2×10 １２ vg / ml, about 1×10 １２ vg / ml to about 3×10 １２ [[ID=3Z]]vg / ml, 2×10[[ID=3Z]] １２ vg / ml to about 4×10 １２ vg / ml, 3×10 １２ vg / ml to about 5×10 １２ vg / ml, 4×10 １２ vg / ml to about 6×10 １２ vg / ml, 5×10 １２ vg / ml to about 7×10 １２ vg / ml, 6×10 １２ vg / ml to about 8×10 １２ vg / ml, 7×10 １２ vg / ml to about 9×10 １２ vg / ml, or 8×10 １２ [[ID=S8]]vg / ml to about 1×10 １３ The composition according to claim 1 or 2, comprising a dosage selected from the group of from 8×10

11. The composition according to claim 1 or 2, wherein the expression vector is selected from plasmids, liposomes, lentiviral vectors, adenovirus vectors, or adeno-associated vectors (AAVs).

12. A composition for use in a method for delivering an expression vector to a ventromedial nucleus of a target requiring the expression vector, wherein the method is (a) Injection catheter having a step distance of approximately 0.5 mm to approximately 2.0 mm from the injection tip; (b) Infusion rate of approximately 0.001 ml / min to approximately 0.015 ml / min; (c) an injection volume of approximately 250 μl to approximately 750 μl per hemisphere, wherein the injection is performed at approximately 3 to 4 injection sites; and (d) Approximately 3 x 10 １１ vg / ml ~ approx. 1×10 １３ vg / ml dosage This includes the injection of the vector by A composition in which the delivery avoids one or more of the anterior hippocampal crust, parahippocampal crust, hippocampal crust, or hippocampal region.

13. The composition according to claim 12, wherein the subject is suffering from cognitive impairment.

14. The composition according to claim 12 or 13, wherein the expression vector further comprises a therapeutic polynucleotide.

15. The composition according to claim 14, wherein the polynucleotide encodes brain-derived neurotrophic factor (BDNF).

16. The aforementioned administration was approximately 3 × 10 １１ vg / ml ~ approx. 5 x 10 １１ vg / ml, approximately 4×10 １１ vg / ml ~ approx. 6×10 １１ vg / ml, approximately 5×10 １１ vg / ml ~ approx. 7×10 １１ vg / ml, 6 x 10 １１ vg / ml ~ approx. 8 x 10 １１ vg / ml, approximately 7×10 １１ vg / ml ~ approx. 9 x 10 １１ vg / ml, approximately 8×10 １１ vg / ml ~ approx. 1×10 １２ vg / ml, approximately 9×10 １１ vg / ml ~ approx. 2×10 １２ vg / ml, approximately 1×10 １２ vg / ml ~ approx. 3 x 10 １２ vg / ml, 2 x 10 １２ vg / ml ~ approx. 4 x 10 １２ vg / ml, 3 x 10 １２ vg / ml ~ approx. 5 x 10 １２ vg / ml, 4 x 10 １２ vg / ml ~ approx. 6×10 １２ vg / ml, 5 x 10 １２ vg / ml ~ approx. 7×10 １２ vg / ml, 6 x 10 １２ vg / ml ~ approx. 8 x 10 １２ vg / ml, 7 x 10 １２ vg / ml ~ approx. 9 x 10 １２ vg / ml, or 8 x 10 １２ vg / ml ~ approx. 1×10 １３ The composition according to claim 12 or 13, comprising a dose selected from the group vg / ml.

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