Gene therapy for treatment of cognitive disorders

Abstract

Provided herein are methods and compositions for treating cognitive disorders.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 63 / 328,934, filed on April 8, 2022, the contents of which are incorporated herein by reference in their entirety. Government Support Statement

[0002] This invention was made with government support under Grants AG043416 and AG010435 from the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0003] The present disclosure generally relates to the field of methods for accurately 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 the network of this brain region, including the exact anatomical target, vector concentration, and vector volume. Summary of the invention

[0004] One embodiment of the present disclosure relates to a method for improving cognitive function in a subject in need thereof, the method comprising or alternatively consisting essentially of or further consisting of the following steps: 11 vg / ml to about 1×10 13 vg / ml dose to the ventromedial nucleus of the subject encoding a therapeutic peptide or a polynucleotide of brain-derived neurotrophic factor (BDNF), the dose is administered at an infusion rate of about 0.001ml / minute to about 0.015ml / minute and an infusion volume of about 250μl to about 750μl per hemisphere or alternatively per two hemispheres, thereby improving the cognitive function of the subject. Non-limiting examples of cognitive function include short-term memory or long-term memory, or various aspects of dementia. Other diseases and disorders are known in the art and are described herein. Methods for determining improvement are known in the art. Compared to a previous time point or for an average measurement of a group of subjects who may or may not suffer from the same injury, the improvement of each individual subject can be measured. In one aspect, the method also includes or is essentially composed of or is further composed of the following steps: determining cognitive function before or after administering treatment, and optionally comparing the test results with baseline values. The determination may include a set of appropriate biomarkers or other physical parameters or clinical parameters.

[0005] In some embodiments, the subject has a disorder selected from Alzheimer's disease (AD), mild cognitive impairment, presymptomatic AD, frontotemporal dementia, or dementia with Lewy bodies.

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

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

[0008] In some embodiments, the polynucleotide also comprises an expression vector, and the polynucleotide is administered in the expression vector. Non-limiting examples of such vectors include, for example, plasmids, liposomes, lentiviral vectors, adenoviral vectors, or adeno-associated vectors (AAV). Methods for preparing such vectors are known in the art and are briefly described herein. In some embodiments, the polynucleotide is operably linked to a regulatory nucleotide to drive the expression of the polynucleotide. Non-limiting examples of such regulatory nucleotides include promoters and enhancer elements.

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

[0010] In some embodiments, the administration does not involve or excludes one or more of the following: substantial delivery to a selected area of ​​the brain selected from one or more of the presubiculum, parasubiculum, subiculum, or hippocampus. In another aspect, the administration does not involve or excludes substantial delivery to a selected area of ​​the brain selected from two or more, three or more, or all of the presubiculum, parasubiculum, subiculum, or hippocampus.

[0011] In some embodiments, the polynucleotide is administered to a subject at 3 or 4 infusion sites.

[0012] In some embodiments, the drug is administered in one or more doses, and each dose contains at least 3×10 11 vg / ml. In some embodiments, one or more doses are administered, and the amount of each dose is selected from: about 3×10 11 vg / ml to about 5×10 11 vg / ml, about 4×10 11 vg / ml to about 6×10 11vg / ml, about 5×10 11 vg / ml is about 7×10 11 vg / ml, 6×10 11 vg / ml is about 8×10 11 vg / ml, about 7×10 11 vg / ml is 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 is about 2×10 12 vg / ml, about 1×10 12 vg / ml to about 3×10 12 vg / ml, 2×10 12 vg / ml is 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 is about 7×10 12 vg / ml, 6×10 12 vg / ml is about 8×10 12 vg / ml, 7×10 12 vg / ml is about 9×10 12 vg / ml, or 8×10 12 vg / ml to about 1×10 13 vg / ml. In another aspect, provided herein are compositions comprising a specific dose of a polynucleotide and / or vector having the above vg / ml. The composition may also comprise a preservative or cryoprotectant or other agent for ease of delivery. In another aspect, the composition is lyophilized.

[0013] One embodiment of the present disclosure is directed to a method for delivering an expression vector to the ventromedial nucleus of a subject in need thereof, the method comprising or consisting essentially of or further consisting of the following steps: administering the vector by infusion of a vector comprising or consisting essentially of or further consisting of: (a) an infusion catheter having a step distance from the infusion tip of about 0.5 mm to about 2.0 mm; (b) an infusion rate of about 0.001 ml / min to about 0.015 ml / min; (c) an infusion volume of about 250 μl to about 750 μl per hemisphere, wherein the infusion occurs between about 3 to about 4 infusion sites; and (d) about 3×10 11 vg / ml to about 1×10 13vg / ml, and wherein the delivery avoids the pre-subiculum, para-subiculum, subiculum or hippocampus region, and the subject suffers from cognitive impairment. In one aspect, the expression vector is delivered via an infusion cannula having a stepped design with a distance of 0.5 mm to 2.0 mm from the infusion tip.

[0014] In some embodiments, the expression vector also comprises a therapeutic polynucleotide. In some embodiments, the polynucleotide encoding is selected from the following proteins: brain-derived neurotrophic factor (BDNF), palmitoyl-protein thioesterase 1 (PPT1), tripeptidyl peptidase 1, CLN6 (Linclin), CLN8, cathepsin D or MFSD8 or battenin. Other therapeutic proteins with neurological function are known in the art. In one aspect, the expression vector is delivered via an infusion cannula having a step design at a distance of 0.5mm to 2.0mm from the infusion tip.

[0015] In some embodiments, the expression vector is selected from a plasmid, a liposome, a lentiviral vector, an adenoviral vector or an adeno-associated vector (AAV). Methods for preparing such vectors are known in the art and are briefly described herein. In some embodiments, the polynucleotide is operably linked to a regulatory nucleotide to drive the expression of the polynucleotide. Non-limiting examples of such regulatory nucleotides include promoters and enhancer elements.

[0016] In some embodiments, the drug is administered in one or more doses, and each dose contains at least 3×10 11 vg / ml. In some embodiments, one or more doses are administered, and the amount of each dose is selected from: 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 is about 7×10 11 vg / ml, 6×10 11 vg / ml is about 8×10 11 vg / ml, about 7×10 11 vg / ml is 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 is about 2×10 12 vg / ml, about 1×10 12 vg / ml to about 3×10 12 vg / ml, 2×1012 vg / ml is 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 is about 7×10 12 vg / ml, 6×10 12 vg / ml is about 8×10 12 vg / ml, 7×10 12 vg / ml is about 9×10 12 vg / ml, or 8×10 12 vg / ml to about 1×10 13 In one aspect, the expression vector is delivered via an infusion cannula having a stepped design with a distance of 0.5 mm to 2.0 mm from the infusion tip.

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

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

[0019] Also provided are kits comprising compositions for practicing the methods as described herein, optionally including instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Schematic diagram of the entorhinal cortex and its major afferent and efferent connections to the hippocampus and cerebral cortex. The entorhinal cortex (layers 1.1-1.1.1) projects primarily to the outer molecular layer of the dentate gyrus (DG) and to area CM of the hippocampus. Area CAI 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] . Reproduced from Nagahra et al. (2018) Gene Therapy (2018) vol. 25: pp. 104-114.

[0021] Figures 2A to 2H: Location of the rhesus monkey entorhinal cortex (EC) and its corresponding MR images. Schematic representation of the entorhinal cortex across four coronal planes (adapted from Paxinos et al.

[34] ), which are located from –2.7 mm to –14.85 mm from bregma (Fig. 2A, Fig. 2C to Fig. 2E). The entorhinal cortex is located on the ventral and medial surfaces of the temporal lobe, with local landmarks including the olfactory fissure (RF), perirhinal cortex (PR), amygdala (Am), subiculum (S), and hippocampus (Hp). T1 and T2 MRI scans (Fig. 2B, Fig. 2F to Fig. 2H), with visible landmarks of the olfactory fissure (RF) indicated on the T2 images (arrows in Fig. 2A, Fig. 2B, Fig. 2D, Fig. 2G). Reproduced from Nagahra et al. (2018) Gene Therapy (2018) vol. 25: pp. 104–114.

[0022] Figures 3A to 3F: Real-time MRI scans of the delivery of AAV2-BDNF into the entorhinal cortex (entorhinal cortex) of non-human primates (Figures 3A to 3C), achieving accurate targeted BDNF delivery (Figures 3D to 3F). (Figure 3A) There is an MR-compatible needle that passes through the cortex and striatum (triangle) to reach the ventral and medial entorhinal cortex (arrows). The spread of gadoteridol at the infusion site can be seen (arrows). The inset shows the gadoteridol signal at a higher magnification. (Figure 3B) Matched tissue sections from the same animal show BDNF diffusion in the same area predicted by MR imaging by immunolabeling. (Figure 3C) MR shows the diffusion pattern of gadolinium within the entorhinal cortex of different subjects, and d matching tissue sections. (Figure 3E) MR shows the spread of the vector in a third subject, where gadolinium diffuses along the cortical surface (arrows), and f matching BDNF immunolabeled sections. Arrows indicate bands of layer II entorhinal cells containing BDNF. Hp hippocampus. Scale bar, (Fig. 3D) = 0.5 mm, (Fig. 3E, Fig. 3F) = 1 mm. Reproduced from Nagahra et al. (2018) Gene Therapy (2018) Vol. 25: pp. 104-114.

[0023] Figures 4A to 4D: AAV2-BDNF and AAV2-GFP predominantly transduce neurons. At the infusion site, (Figure 4A) BDNF immunolabeling and (Figure 4B) GFP immunolabeling were observed predominantly in (Figure 4C) neurons labeled with NeuN. (Figure 4D) Overlap. When quantified, 88.2±3.8% of GFP-expressing cells were co-labeled with NeuN. Scale bar = 50 pm. Reproduced from Nagahra et al. (2018) Gene Therapy (2018) Vol. 25: pp. 104-114.

[0024] Figures 5A to 5E: Distribution of BDNF-labeled neurons in the entorhinal infusion site, (Figure 5A) 13DNF immunolabeling in the AAV2-BDNF infusion site in the entorhinal cortex. (Figure 5B) Single cell profile of BDNF immunolabeling, and (Figure 5C) Area of ​​BDNF-containing cells used to quantify vector volume distribution. (Figure 5D), (Figure 5E) The volume of AAV2-BDNF vector infused was significantly correlated with the tissue volume containing BDNF-labeled neurons (p < 0.001) and the number of BDNF-labeled neurons (p < 0.001). Reprinted from Nagahra et al. (2018) Gene Therapy (2018) Vol. 25: pp. 104-114.

[0025] Figures 6A to 6D: BDNF diffuses into the hippocampus. (Figure 6A) BDNF immunoreactivity in the hippocampus of a control subject shows endogenous expression of BDNF in the mossy fiber terminal regions of the CA3 lucidum and hilus regions. (Figure 6B) After infusion of AAV2-BDNF into the entorhinal cortex, BDNF immunoreactivity is visible in the outer molecular layer of the hippocampus (triangle). (Figure 6C) Fluorescent labeling shows BDNF immunoreactive fibers in the outer molecular layer (OML) of the hippocampus, but not in its inner molecular layer (IML) or granule cell layer (GC). (Figure 6D) The scatter plot shows that even smaller volumes of AAV2-BDNF infused into the entorhinal cortex can lead to widespread increased (approximately 70%) BDNF expression in the OML of the dentate gyrus. Scale bars: (Figure 6A, Figure 6B) 1 mm; (Figure 6C) 200 μm. Reproduced from Nagahra et al. (2018) Gene Therapy (2018) 25: 104-114.

[0026] Figures 7A to 7C show the urgent need to target the entorhinal subregion to effectively and safely treat memory disorders. These figures show 1×10 12 vg / ml to 1×10 13 vg / ml (see Table 2) and suggests that this dose is tolerated when precisely targeting the entorhinal cortex. These titers were as high as 10 13 Injection of 100 vg / ml resulted in good entorhinal expression without the toxicity (seizures) associated with higher doses or vector mistargeting. The triangle below indicates the area of ​​BDNF gene expression after vector infusion. (Fig. 7A) Monkey 21684. (Fig. 7B) Monkey 22241. (Fig. 7C) Monkey 24647.

[0027] Figures 8A to 8E: The figures show that, in contrast, the following infusions restricted to the entorhinal cortex 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] 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 areas 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] Fig. 10A-10B: A novel infusion cannula design with a stepped 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 infusion tip. (Fig. 10A) Prior art design. (Fig. 10B) Prior art design. DETAILED DESCRIPTION definition

[0030] The following will more fully describe embodiments according to the present disclosure. However, various aspects of the present disclosure can be embodied in different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to make the present disclosure 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 herein are only used for the purpose of describing specific embodiments and are not intended to be limiting.

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

[0032] The terms used in the description herein are for the purpose of describing particular embodiments only and are not intended to limit the present 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 indicates otherwise, it is specifically intended that the various features of the present disclosure described herein may be used in any combination. In addition, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification indicates that a composite comprises components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted and abandoned, either individually or in any combination.

[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 symbols (e.g., pH, temperature, time, concentration, and molecular weight, including ranges) are approximate and, where appropriate, vary positively (+) or negatively (-) in increments of 1.0 or 0.1, or alternatively by + / -15%, or alternatively by 10%, or alternatively by 5%, or alternatively by 2%. It should be understood that, although not always expressly stated, all numerical symbols are preceded by the term "about". It should also be understood that, although not always expressly stated, the agents described herein are exemplary only and that equivalents of such agents are known in the art.

[0037] Throughout the disclosure, various publications, patents, and published patent specifications are referenced by identifying citations or by Arabic numerals. The full citation for a publication identified by an Arabic numeral can be found immediately preceding the claims. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference in their entirety into the present disclosure in order to more fully describe the state of the art to which the present disclosure relates.

[0038] The practice of the present disclosure will employ, 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); "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 the present disclosure and the appended claims, the singular forms "a," "an," "the," and "said" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

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

[0041] These terms or "acceptable," "effective," or "sufficient" when used to describe the selection of any component, range, dosage form, etc. disclosed herein, mean that the component, range, dosage form, etc. is suitable for the disclosed purpose.

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

[0043] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the virus class associated with the name and belonging to the genus dependoparvovirus of the family Parvoviridae. A variety of serotypes of the virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 AAV serotypes numbered sequentially are known in the art. Non-limiting exemplary serotypes for use in the methods disclosed herein include any of the 11 serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAVPHP.B. AAV particles contain or alternatively consist essentially of or are further composed of three major viral proteins: VP1, VP2, and VP3. In one embodiment, AAV refers to serotype 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 further consist of all kingdoms of life except the prokaryotes. They can be easily distinguished by membrane-bound nuclei. Animals, plants, fungi and protists are eukaryotic organisms or organisms whose cells are organized into complex structures by endomembranes and cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise specifically listed, 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 apes, cattle, pigs, mice, rats, birds, reptiles and humans, such as HEK293 cells and 293T cells.

[0045] "Prokaryotic cells" generally lack a nucleus or any other membrane-bound organelles, and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells may also store genetic information in circular loops called episomes. Bacterial cells are very small, about the size of animal mitochondria (about 1μm to 2μm in diameter and 10μm long). Prokaryotic cells have three main shapes: rod-shaped, spherical, and spiral. Instead of going through a complex replication process like eukaryotic organisms, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus bacteria, E. coli bacteria, and Salmonella bacteria.

[0046] Unless otherwise indicated, it is not necessary to explicitly state that when the present disclosure relates to polypeptides, proteins, polynucleotides or antibodies, their equivalents or biological equivalents will also be within the scope of the present disclosure. As used herein, when referring to a reference protein, antibody, polypeptide or nucleic acid, the term "biological equivalent thereof" is intended to be synonymous with "equivalent thereof", referring to a protein, antibody, polypeptide or nucleic acid having minimum sequence identity while still maintaining the desired structure or function. Unless specifically listed herein, any polynucleotide, polypeptide or protein mentioned herein is considered to also include its equivalent. For example, an equivalent means having at least about 70% homology or identity, or at least about 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 percentage with a reference protein, polypeptide or nucleic acid over the entire length of the reference sequence, and exhibiting a biological activity substantially equivalent to that of a reference protein, polypeptide or nucleic acid. Alternatively, when referring to a polynucleotide, its equivalent is, in one aspect, a polynucleotide that hybridizes to a reference polynucleotide or its complement under stringent conditions, and its equivalent has the same or similar activity or function as the reference polynucleotide or its complement in another aspect.

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

[0048] The term "encoding" when applied to a nucleic acid sequence means that if a polynucleotide can be transcribed and / or translated to produce mRNA of a polypeptide and / or its fragments in its native state or when manipulated by methods well known to those skilled in the art, then the polynucleotide is said to "encode" a polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0049] When referring to a specific molecule, organism or cell material, the term "equivalent" or "biological equivalent" is used interchangeably and refers to a specific molecule, organism or cell material with minimal homology while still maintaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides with at least 60%, or alternatively at least 65%, or alternatively 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 with it or a polypeptide sequence, or a polypeptide encoded by a polynucleotide or its complement, which hybridizes with a polynucleotide encoding such a polypeptide sequence under high stringency conditions. High stringency conditions are as described herein and are incorporated herein by reference. Alternatively, an equivalent thereof 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 with 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 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 97% identity with a reference polynucleotide. Equivalents also refer to 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, when comparing two sequences, the percentage of bases (or amino acids) is the same. Such alignments and homology or sequence identity percentages can be determined using software programs known in the art, such as those described in Appendix 30, Section 7.7.18, Table 7.7.1 of "A Manual of Molecular Biology" (Ausubel et al., ed., 1987). In certain embodiments, the alignment is performed using default parameters. 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; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + 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 identity percentage can be determined by incorporating them into clustalW (available at: genome.jp / tools / clustalw / , last accessed on January 13, 2017).

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

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

[0054] "Hybridization" refers to the reaction of one or more polynucleotides to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex may comprise or alternatively consist essentially of or further consist of the following items: two chains forming a duplex structure, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination of these substances. The hybridization reaction may constitute a step in a broader 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 6×SSC to about 10×SSC; a formamide concentration of about 0% to about 25%; and a washing solution of about 4×SSC to about 8×SSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9×SSC to about 2×SSC; a formamide concentration of about 30% to about 50%; and a washing solution of about 5×SSC to about 2×SSC. Examples of high stringency conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1×SSC to about 0.1×SSC; a formamide concentration of about 55% to about 75%; and a washing solution of about 1×SSC, 0.1×SSC, or deionized water. Typically, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2 or more washing steps, and the wash incubation time is about 1 minute, 2 minutes, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It will be appreciated that the equivalent of SSC using other buffer systems may be employed.

[0056] As used herein, "expression" refers to the process of polynucleotide transcription into mRNA and / or the subsequent translation of transcribed mRNA into peptides, polypeptides or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing mRNA in eukaryotic cells.

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

[0058] "Under transcriptional control" is a term well known in the art and means that the transcription of a polynucleotide sequence (usually a DNA sequence) is dependent upon its operative linkage to elements that aid in initiating or promoting transcription. "Operably linked" means that polynucleotides are arranged in a manner that allows them to function in a cell.

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

[0060] As used herein, the term "functional" may be applied to the modification of any molecule, biological product, or cellular material in order to achieve a particular, specific effect.

[0061] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising or alternatively consisting essentially of or further consisting of purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0062] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence (such as 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 the start and rate of transcription control in a polynucleotide sequence. It can contain genetic elements that can bind regulatory proteins and molecules, such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include Rous sarcoma virus (RSV) LTR promoters (optionally with RSV enhancers), cytomegalovirus (CMV) promoters, SV40 promoters, dihydrofolate reductase promoters, β-actin promoters, phosphoglycerol kinase (PGK) promoters, U6 promoters or EF1 promoters. In some embodiments, the promoter is chicken β-actin ("CBA") promoter.

[0063] Other non-limiting exemplary promoters with certain 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 α-1-antitrypsin. Synthetic derived promoters can be used for ubiquitous or tissue-specific expression. In addition, viral derived promoters, a portion of which as described above, can be used in the methods disclosed herein, such as CMV, HIV, adenovirus and AAV promoters. In some embodiments, the promoter is coupled to an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include RSV enhancers or CMV enhancers.

[0064] Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer / promoter is an enhancer / promoter that is naturally connected to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is an enhancer / promoter that is juxtaposed to a gene by genetic manipulation (i.e., molecular biology techniques) so that the transcription of the gene is directed by the connected enhancer / promoter.

[0065] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains or alternatively consists essentially of or further consists of a complete replicon so that the vector can be replicated when placed in a cell, for example, by a transformation process. The vector can be a viral vector or a non-viral vector. Viral vectors include retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papillomaviruses, or other modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles that contain or alternatively consist essentially of or further consist of DNA condensed with cationic polymers, such as heterologous polylysine, oligopeptides of limited length, and polyethyleneimine, in some cases contained in liposomes; and use a ternary complex that contains or alternatively consists essentially of or further consists of a virus and polylysine-DNA.

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

[0067] "Gene delivery vehicle" is defined as any molecule that can carry the inserted polynucleotide into the host cell. Examples of gene delivery vehicles are liposomes, micelles, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria or viruses (such as baculovirus, adenovirus and retrovirus), phage, cosmids, plasmids, fungal vectors and other recombinant vectors commonly used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as for simple protein expression.

[0068] Gene delivery vehicles can be used to deliver the polynucleotides disclosed herein to cells or tissues. "Gene delivery", "gene transfer", "transduction", etc., as used herein, refer to the term for introducing exogenous polynucleotides (sometimes referred to as "transgenic") into host cells, which is independent of the method used for introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (through, for example, viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), and technologies that promote the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery, and various other technologies for the introduction of polynucleotides). The introduced polynucleotides can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides either contain a replication origin compatible with the host cell, or are integrated into the host cell's replicon such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Many known vectors are capable of mediating the transfer of genes to mammalian cells, as known in the art and described herein.

[0069] "Plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population and often provide a selective advantage under given environmental conditions. Plasmids can carry genes that provide resistance to naturally occurring antibiotics in competitive environmental niches, or alternatively, the proteins produced can function as toxins under similar environments.

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

[0071] In the aspect of gene transfer mediated by DNA viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide comprising or alternatively consisting essentially of or further consisting of a viral genome or a portion thereof and a transgene. Adenovirus (Ad) is a relatively well-characterized homogeneous virus group, including more than 50 serotypes. Ad does not need to be integrated 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 viruses. Such vectors are commercially available from sources such as Takara Bio USA (Mountain View, CA), VectorBiolabs (Philadelphia, PA) and Creative Biogene (Shirley, NY). Wild-type AAV has high infectivity and specificity for integration into the host cell genome. See Wold and Toth (2013) Curr. Gene. Ther. Vol. 13 No. 6: pp. 421-433, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, Vol. 81: pp. 6466-6470, and Lebkowski et al. (1988) Mol. Cell. Biol. Vol. 8: pp. 3988-3996.

[0072] The vectors containing both promoter and polynucleotide operably connected to the cloning site are well known in the art. Such vectors can transcribe RNA in vitro or in vivo, and can be commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or change the 5' and / or 3' non-translated portion of the clone to eliminate additional, potential inappropriate alternative translation initiation codons or other sequences that may interfere or reduce expression at the transcription or translation level. Alternatively, a common ribosome binding site can be directly inserted into the 5' of the start codon to enhance expression.

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

[0074] As used herein, the term "signal peptide" or "signal polypeptide" means an amino acid sequence that is typically present at the N-terminus of a newly synthesized secretory or membrane-bound polypeptide or protein. It is used to direct the polypeptide to a specific cellular location, such as across a cell membrane, into a cell membrane, or into a cell 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. Patent Nos. 8,853,381, 5,958,736, and 8,795,965.

[0075] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or capsid of a viral particle. The function of the capsid is to encapsidate, protect, transport and release the viral genome into a host cell. The capsid is usually composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidation" means being contained within a viral capsid.

[0076] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid that is used to provide other components necessary for replication and packaging of viral particles or recombinant viral particles (such as the modified AAV disclosed herein). The components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication and / or packaging. For example, the helper virus may encode enzymes necessary for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus) or herpes virus (virus).

[0077] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infected helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press (New York). It is fully expected that the same principles described in these reviews will apply to other AAV serotypes characterized after the publication date of these reviews, because it is well known that various serotypes are very closely related in structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, edited by JR Pattison; and Rose, Comprehensive Virology, Vol. 3: pp. 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins, such as those expressed in AAV2. The extent of relatedness is further indicated by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication function of each serotype is under similar regulation.

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

[0079] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle consisting of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises or alternatively consists essentially of or further consists of a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as an "AAV vector particle" or simply "AAV vector". Therefore, the production of AAV vector particles necessarily includes the production of AAV vectors, because such vectors are contained within the AAV vector particles.

[0080] In some embodiments, AAV is a replication-deficient parvovirus with a single-stranded DNA genome of about 4.7 kb in length, comprising two 145-nucleotide inverted terminal repeats (ITRs). AAV has multiple serotypes. 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 Srivastava et al., J. Virol., Vol. 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. AF085716. The genome is provided in GenBank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., Vol. 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., Vol. 13 No. 1: 67-76 (2006); and the AAV-11 genome is provided in Virology, Vol. 330 No. 2: 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928, which is incorporated herein by reference. U.S. Pat. No. 9,434,928 also provides the sequences of the capsid protein and the self-complementary genome. In one aspect, the genome is a self-complementary genome. The cis-acting sequences for guiding viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained in AAV ITR. Three AAV promoters (named as p5, p19 and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding rep and cap genes. Two rep promoters (p5 and p19) are coupled with the differential splicing (at nucleotides 2107 and 2227) of a single AAV intron, resulting in the production of four rep proteins (rep 78, rep 68, rep 52 and rep 40) from the rep gene. Rep protein has a variety of enzyme properties that are ultimately responsible for replicating viral genomes. The cap gene is expressed from the p40 promoter, and it encodes three kinds of capsid proteins VP1, VP2 and VP3. Alternative splicing and non-shared translation start sites are responsible for the generation of three kinds of related capsid proteins. A single shared 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, Vol. 158: 97-129 (1992).

[0081] AAV has unique characteristics that make it an attractive vector for delivering exogenous DNA to cells, such as in gene therapy. AAV infection of cells in culture is non-cytopathic, while natural infection in humans and other animals is silent and asymptomatic. In addition, AAV infects many mammalian cells, making it possible to target many different tissues in vivo. In addition, AAV transduces slowly dividing cells and non-dividing cells, and can be essentially persistent throughout the life cycle of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted into a plasmid as cloned DNA, which makes it feasible to construct a recombinant genome. In addition, since the signal for directing AAV replication and genome encapsidation is contained in the ITR of the AAV genome, a portion or all of the internal approximately 4.3kb genome (encoding replication and structural capsid proteins, rep-cap) can be replaced by exogenous DNA. In order to produce AAV vectors, rep and cap proteins can be provided in trans. Another notable feature of AAV is that it is an extremely stable and powerful virus. It can easily withstand the conditions used to inactivate adenovirus (56°C-65°C for several hours), making cryopreservation of AAV less critical. AAV can even be lyophilized. Finally, cells infected with AAV are not resistant to repeated infection.

[0082] The recombinant AAV (rAAV) genome of the present disclosure comprises or alternatively consists essentially of or further 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 may be 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, AAVPHP.B, and AAV rh74. The preparation of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, Vol. 22, No. 11: pp. 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 and another compound or composition or active agent (eg, a gene delivery vehicle).

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

[0085] As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, and emulsions (such as oil / water or water / oil emulsions), and various types 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 Edition (Mack Publishing Company, Easton).

[0086] "Subject", "individual" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cattle, sheep, pigs, dogs, cats, farm animals, sports animals, pets, horses and primates, particularly humans. In addition to being used for human treatment, the present disclosure can also be used for veterinary treatment of companion mammals, non-native animals and domesticated animals (including mammals, rodents, etc.). 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 eighteen.

[0087] "Treating" or "treatment" of a disease includes: (1) preventing the disease, i.e., causing clinical symptoms of the disease to not develop in a patient who may be susceptible to the disease but who does not yet experience or show symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. In one aspect, the term "treating" excludes preventing or prophylaxis.

[0088] The term "suffering from" in connection with the term "treating" refers to a patient or individual who has been diagnosed with a disease or is susceptible to a disease.

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

[0090] The term “cognitive function” refers to a variety of mental abilities including, but not limited to, learning, thinking, reasoning, memory, problem solving, decision making, and attention.

[0091] The term "effective amount" as used herein is intended to mean an amount sufficient to achieve the desired effect. In the case of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the disorder in question and the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the case of gene therapy, in some embodiments, the effective amount is an amount sufficient to restore partial or full function of the defective gene of the subject. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to express the gene in the subject. The technician will be able to determine the appropriate amount based on these and other factors.

[0092] In some embodiments, the effective amount will depend on the size and constitution of the application in question. It also depends on the constitution and sensitivity of the subject being targeted and the method used. Those skilled in the art will be able to determine the effective amount based on these and other considerations. The effective amount may include or alternatively consist essentially of or further consist of one or more administrations of the composition according to the embodiment.

[0093] As used herein, the term "administer / administration / administering" is intended to mean the delivery of a substance to a subject, such as an animal or a human. Administration can be achieved continuously or intermittently in one dose throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, and the age, health or sex of the subject being treated. Single or multiple administrations may be performed, with the dosage level and pattern being selected by the treating physician, or in the case of pets and animals, by the treating veterinarian. Suitable dosage formulations and methods for administering medicaments are known in the art. The route of administration may also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, the health status or disease stage of the subject being treated, and the target cells or tissues. 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 that promotes targeted delivery of drugs 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 an extension of the cannula beyond the tip of the needle.

[0095] As used herein, the term "hemispheric" refers to the two distinct cerebral hemispheres of the brain connected by the corpus callosum.

[0096] BDNF (brain-derived neurotrophic factor) is a 27kDa homodimer that is originally derived from the human brain and promotes the growth of spinal sensory neurons. In some embodiments, BDNF supports the survival and growth of sensory neurons, ganglion neurons, dopaminergic neurons, cholinergic neurons, GABAergic neurons, and motor neurons. GenBank M61176 describes the coding sequence (mRNA) of BDNF, and the last access date was April 7, 2022.

[0097] Modes for carrying out the present disclosure

[0098] In a prior study (Nagahra et al., (2018) Gene Therapy, Vol. 25: pp. 104-114), the applicant reported that the brain-derived neurotrophic factor (BDNF) gene delivered to the entorhinal cortex is a candidate for treating Alzheimer's disease (AD) to reduce neurodegeneration associated with memory loss. Precise targeting of the entorhinal cortex in AD is complicated due to the profound atrophic state of this brain region. Using an MRI-guided approach and convection-enhanced delivery, the applicant accurately and consistently targeted delivery of AAV2-BDNF to the entorhinal cortex of non-human primates; 86±3% of the transduced cells in the targeted region colocalized with the neuronal marker NeuN. AAV2-BDNF (3×10 8 vg / pl) infusion volume and the number of BDNF-labeled cells and volume of BDNF immunoreactivity in the entorhinal cortex (mm 3 ) is linearly correlated. BDNF is normally transported from the entorhinal cortex to the hippocampus; Applicants have also found that after delivery of therapeutic BDNF vectors to the entorhinal cortex, BDNF immunoreactivity is elevated in the hippocampus, thereby enabling distribution of the growth factor through key memory circuits.

[0099] In contrast to this earlier research, applicants describe herein specific methods for gene therapy targeting the entorhinal / hippocampal system to treat cognitive impairment or prevent impending cognitive impairment. The methods of the present invention specify a new and previously unknown set of specific parameters required to treat the entorhinal / hippocampal system in humans. These methods can be applied to Alzheimer's disease (AD).

[0100] Targeting the entorhinal cortex to treat cognitive impairment in humans requires gene delivery to a subregion of the entorhinal cortex, namely the ventromedial nucleus, and avoiding the pre-subiculum, para-subiculum, subiculum, and hippocampus. If these parameters are not followed, the treatment will lack efficacy and lead to neurotoxicity, including seizures and risk of death. To the best of the applicant's knowledge, the literature does not disclose the necessity of targeting this subregion of the entorhinal cortex, nor does it disclose the necessity of avoiding vector injection into the pre-subiculum, para-subiculum, subiculum, and hippocampus.

[0101] In one embodiment, the volume of carrier used to target a sufficient volume of the entorhinal cortex for effective human treatment may be in the range of 250 μl to 750 μl per hemisphere, distributed between 3 to 4 infusion sites. To the applicant's knowledge, the existing literature recommends a much lower carrier range of only 2.5 μl to 25 μl per site, which the applicant now knows is ineffective. In other published literature, a carrier dose of 15 μl to 130 μl per site (up to 375 μl per hemisphere) is recommended, but this is also incorrect because the applicant knows that the dose is too low. The applicant's dose is appropriate, and based on the work described herein, infusion of 30 μl to 870 μl per site (360 μl to 2610 μl per hemisphere or alternatively 250 μl to 750 μl) at 3 to 4 sites has been determined to be the most effective.

[0102] The applicants have also identified new vector concentrations that are effective and safe for use in humans. The applicants have found that gene therapy vector doses below 3×10 11 vg / ml is invalid and above 1×10 13 vg / ml may be toxic. To the best of the applicant’s knowledge, the vector doses previously cited in the literature range from 1×10 10 vg / ml to 1×10 15 vg / ml, which the applicant later determined was incorrect. The literature describes only one dose, 3×10 11 vg / ml, and no applicant has now determined that the maximum value is 1×10 13 Thus, the specific parameters of vector concentration have never been reported before and constitute an important approach for successful and safe gene delivery.

[0103] To the best of applicant's knowledge, the literature has not identified optimal infusion rate parameters. Based on new data from animal subjects, in one aspect, applicant defines an optimal vector infusion rate of 1 μl / min to 15 μl / min to achieve adequate vector diffusion and coverage of the intended target. Volumes less than this value will result in inefficient vector distribution in the brain; volumes exceeding this value will cause tissue damage and associated toxicity.

[0104] To the applicant's knowledge, the literature does not specify optimal hardware for vector infusion to achieve adequate vector spread and coverage of the intended target. The applicant now recognizes that the needle required to achieve adequate vector distribution in the target area should have a single widened "step" (outer diameter expansion) located 0.5 mm to 2.0 mm from the infusion tip in order to reduce vector backflow and losses to the infusion tubing.

[0105] In addition to the above points, Applicants now know that these methods are relevant to treating the prodromal state of Alzheimer's disease, mild cognitive impairment, and treating patients at potential risk of developing Alzheimer's disease. Applicants have indications that these methods can be used to treat pre-symptomatic patients whose cognitive function is intact but who are at high risk of developing Alzheimer's disease based on biomarkers such as cerebrospinal fluid studies and brain positron emission tomography imaging.

[0106] Treatment

[0107] Applicants disclose methods of improving cognitive function in a subject in need thereof, comprising administering to a subject approximately 3×10 11 vg / ml to about 1×10 13 vg / ml dose to the ventromedial nucleus of the subject, the polynucleotide encoding brain-derived neurotrophic factor (BDNF), the dose is administered 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, thereby improving the cognitive function of the subject. In another aspect, administration also includes an infusion catheter having a step distance of about 0.5 mm to about 2.0 mm from the infusion tip.

[0108] Also disclosed herein are methods for delivering an expression vector to the ventromedial nucleus of a subject in need thereof, the methods comprising infusing the vector through: (a) an infusion catheter having a step distance from the infusion tip of about 0.5 mm to about 2.0 mm; (b) an infusion rate of about 0.001 ml / min to about 0.015 ml / min; (c) an infusion volume of about 250 μl to about 750 μl per hemisphere, wherein the infusion occurs between about 3 to about 4 infusion sites; and (d) about 3×10 11 vg / ml to about 1×10 13 vg / ml, and wherein the delivery avoids the pre-subiculum, para-subiculum, subiculum or hippocampus regions, and the subject suffers from cognitive impairment. In another aspect, administering further comprises an infusion catheter having a step distance of about 0.5 mm to about 2.0 mm from the infusion 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 suffers from a disease. In some embodiments, the disease is a cognitive disorder. In some embodiments, the cognitive disorder is Alzheimer's disease (AD), mild cognitive impairment, presymptomatic AD, frontotemporal dementia, or Lewy body dementia. In some embodiments, the subject is a subject with presymptomatic and intact cognitive function, but the subject is at high risk of developing AD based on diagnostic biomarkers, including but not limited to cerebrospinal fluid studies and brain positron emission tomography imaging.

[0110] The titer of the expression vector to be administered in the methods of the present disclosure will vary depending on, for example, the specific vector, mode of administration, therapeutic goal, individual, and cell type to be 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 a titer of at least about 3×10 11 vg / ml, about 4×10 11 vg / ml, about 5×10 11 vg / ml, about 6×10 11 vg / ml, about 7×10 11 vg / ml, about 8×10 11 vg / ml, about 9×10 11 vg / ml, about 1×10 12 vg / ml, about 2×10 12 vg / ml, about 3×10 12 vg / ml, about 4×10 12 vg / ml, about 5×10 12 vg / ml, about 6×10 12 vg / ml, about 7×10 12 vg / ml, about 8×10 12 vg / ml, about 9×10 12 vg / ml to about 1×10 13 In some embodiments, the dose of the expression vector is 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 is about 7×10 11 vg / ml, 6×10 11 vg / ml is about 8×10 11 vg / ml, about 7×10 11 vg / ml is about 9×1011 vg / ml, about 8×10 11 vg / ml to about 1×10 12 vg / ml, about 9×10 11 vg / ml is about 2×10 12 vg / ml, about 1×10 12 vg / ml to about 3×10 12 vg / ml, 2×10 12 vg / ml is 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 is about 7×10 12 vg / ml, 6×10 12 vg / ml is about 8×10 12 vg / ml, 7×10 12 vg / ml is about 9×10 12 vg / ml, or 8×10 12 vg / ml to about 1×10 13 In some embodiments, the dose is at least 3×10 11 In another aspect, administering further comprises an infusion catheter having a step distance from the infusion tip of about 0.5 mm to about 2.0 mm.

[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. Infusion rate can be at least about 0.001ml / minute, about 0.0015ml / minute, about 0.002ml / minute, about 0.0025ml / minute, 0.003ml / minute, about 0.0035ml / minute, 0.004ml / minute, about 0.0045ml / minute, 0.005ml / minute, about 0.0055ml / minute, 0.006ml / minute, about 0.0065ml / minute, 0.007ml / minute, about 0.0075ml / minute, 0.008ml / minute, about 0.0085ml / minute, 0.009ml / minute, about 0.0095ml / minute, about 0.01ml / minute to about 0.015ml / minute in the scope of minute. In another aspect, use also comprises the infusion catheter with the step distance of about 0.5mm to about 2.0mm from infusion tip.

[0112] In some embodiments, any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein are administered with a dose infusion volume of about 250 μl to about 750 μl per hemisphere of the brain. The infusion volume may be 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 infusion volume is administered at about 3 to about 4 infusion sites. In another aspect, administering further comprises an infusion catheter having a step distance from about 0.5 mm to about 2.0 mm from the infusion 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 another aspect, administration further comprises an infusion catheter having a step distance from the infusion tip of about 0.5 mm to about 2.0 mm.

[0114] In some embodiments, the methods disclosed herein include topical administration of any of the polynucleotides, plasmids, viral vectors, or compositions disclosed herein. In some embodiments, the methods disclosed herein include applying 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, epithelium, connective, and neural 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, administration avoids the anterior subiculum, 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, 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 may be 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 the loss on the reflux and infusion pipeline.

[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 regulatory nucleotide. In some embodiments, the expression vector is a lentiviral vector, an adenoviral vector, or an adeno-associated vector (AAV).

[0117] In another aspect, administering further comprises an infusion catheter having a step distance from about 0.5 mm to about 2.0 mm from the infusion tip.

[0118] Applicants have indications that these methods could be used to treat pre-symptomatic patients whose cognitive function is intact but who are at high risk of developing Alzheimer's disease, based on biomarkers such as cerebrospinal fluid studies and brain positron emission tomography imaging.

[0119] It will be apparent to one skilled in the art that the above methods and compositions can be combined with other therapeutic compositions and agents for use in treating or the disclosed diseases or conditions.

[0120] Methods for preparing AAV vectors

[0121] Methods for preparing adeno-associated virus (AAV) vectors (e.g., viruses or viral particles) are disclosed herein, and complementary methods for preparing AAV vectors are known in the art. For example, such methods are disclosed in, for example, WO 01 / 83692, which is incorporated herein by reference in its entirety. The general principles of AAV production are reviewed in, for example, Carter, Current Opinions in Biotechnology, pp. 1533-1539, 1992; and Muzyczka, Curr. Topics in Microbial. and Immunol. Vol. 158: pp. 97-129, 1992, each of which is incorporated herein by reference in its entirety. Various methods for producing AAV are described in Ratschin et al., Mol. Cell. Biol. Vol. 4: p. 2072, 1984; Hermonat et al., Proc. Natl. Acad. Sci. USA, Vol. 81: p. 6466, 1984; Tratschin et al., Mol. Cell. Biol. Vol. 5: p. 3251, 1985; McLaughlin et al., J. Virol., Vol. 62: p. 1963, 1988; and Lebkowski et al., Mol. Cell. Biol., Vol. 7: p. 349, 1988; Samulski et al., J. Virol., Vol. 63: pp. 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, Vol. 13: pp. 1244-1250, 1995; Paul et al., Human Gene Therapy, Vol. 4: pp. 609-615, 1993; Clark et al., Gene Therapy, Vol. 3: pp. 1124-1132, 1996; U.S. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595, each of which is incorporated herein by reference in its entirety. In some embodiments, the 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 embodiments, the cell is a recombinant cell that stably expresses adeno-associated virus rep and cap genes. In some embodiments, the method also includes culturing the cell to produce a transduced cell population. In some embodiments, the method also includes collecting supernatant from the transduced cell population. In some embodiments, the method also includes performing one or more purification steps on the supernatant to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cell debris and protein. Alternatively or additionally, the method also includes lysing the transduced cell population to produce a cell lysate. In some embodiments, the method also includes performing one or more purification steps on the cell lysate to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cell debris and protein. In some embodiments, 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 a viral packaging system (such as a retrovirus, adenovirus, herpes virus or baculovirus packaging system). In some embodiments, packaging is achieved by using a helper virus or a helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that facilitate the delivery of genetic material to the cell. In another aspect, the helper plasmid or a polynucleotide comprising or alternatively consisting essentially of or further consisting of the helper plasmid is stably integrated into the genome of the packaging cell line so that the packaging cell line does not need to be additionally transfected with a helper plasmid.

[0123] Vector production and preparation

[0124] Prior art methods can be used for vector production. As disclosed in Nagahara et al. (2018) and reproduced herein, the AAV2-BDNF vector was generated by UNC Vector Core (University of North Carolina). The vector genome consists of human BDNF cDNA with a CAG promoter, which consists of a human cytomegalovirus (CMV) enhancer, a chicken 13-actin promoter and a splice donor, an intron, and a rabbit fl-globin splice acceptor. The vector was designed to be similar to the AAV2 vectors used in phase 1 and 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 expressed by C s Cl 2 The AAV2-BDNF vector was purified by centrifugation, FPLC and sterile filtration. 12 vg / ml and aliquoted into 100 μl volumes. For infusion, AAV2-BDNF was delivered at 3 × 10 11 vg / ml. The MR contrast agent gadoteridol (Prohance, Bracco Diagnostic, Princeton, NJ) was mixed into the vehicle at a final concentration of 1 mM. A subgroup of subjects (N=9) also received 0.03×10 12 vg / ml titer of AAV2-GFP to confirm the spread of the viral vector in the transduced cells.

[0125] Surgical exposure

[0126] Prior art methods with improvements disclosed by applicants can be used for surgical methods. As disclosed in Nagahara et al. (2018) and reproduced herein, after induction of anesthesia, the animal's head is placed in a stereotactic frame and flexed in a prone position. The head is shaved and cleaned with Nolvasan solution and alcohol. A sterile field is created and a midline incision is made in the skin, muscle, and fascia using electrocautery (Surgistat Electrosurgery, Valleylab, Inc., Boulder, CO). Gently retracting the fascia and muscle exposes the skull over the cortical entry site. Small drill holes are made according to stereotactic coordinates to expose the dura mater over each intended infusion site. The infusion system consisted of a CO “ball joint” array (Hayes Manufacturing Services, USA) fixed to the skull via titanium screws above the craniotomy incision, and (ii) 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] consisted of an 8 cm to 25 cm ceramic section (outer diameter (OD) 1.68 mm) for the main part of the shaft and an 18 mm fused silica section that tapered to an OD of 0.7 mm. The final 3 mm section was a fine fused silica lip (OD 0.36 mm). Briefly, the cannula was connected to a loading line containing the infusate, and the flow rate was regulated using a 1 ml syringe mounted on an MRI-compatible infusion pump. The target was selected and the optimal trajectory was established on the animal's baseline MRI images using neuronavigation software. The distance from the target to the top of the guide rod is then determined in quartz and a depth stop is fixed at the insertion distance in the cannula.After determining the optimal injection parameters, the cannula is manually inserted through the guide rods of the array to the target.

[0127] MRI-guided infusion procedure

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

[0129] Animals were sedated, cannulated, and an intravenous line was established with a 22-gauge to 24-gauge catheter placed in the cephalic or saphenous vein, delivering isotonic fluid at a rate of 5 ml / kg / hour to 10 ml / kg / hour. Isoflurane inhalation anesthesia (Aerrane, Omeda PPD, Liberty, NI) was delivered at 1% to 3% to maintain a stable level of anesthesia. Then, after being placed in a supine position in an MRI-compatible stereotaxic frame, two burr holes were placed for implantation of a spherical joint array consisting of a base (attached to the skull) and a guide tube array (cylinder with three holes) [Salegio et al., Methods Mol Biol. 2016. ]. The monkey was then transferred to the MRI scanner suite. The infusion needle consisted of a ceramic quartz anti-reflux cannula with a 1 mm step at the distal end to prevent vector reflux along 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-inch high-pressure intravenous tubing. The vector was adjusted to a final concentration of 3.0 × 10 11 In nine subjects, one-tenth of the vector infusion volume consisted of AAV2-GFP (3 × 10 11 vg / ml), and 90% of the vector was composed of AAV2-BDNF (3×10 11vg / ml); these monkeys enable analysis of cell types in the brain transduced by the virus. In the initial subjects, the infusion needle was placed in a skull-based stereotaxic frame and advanced into the brain to a point calculated to be located in the middle of the striatum; the accuracy of the needle trajectory and depth was then confirmed with a T2 scan. Because none of these intermediate track scans required correction of the needle trajectory, all subsequent subjects lowered the infusion needle into the entorhinal cortex itself, stopping at a point 1 mm dorsal to the expected target area calculated based on the initial MRI scan. Then, a set of T1, MP-RAGE, and T2 weighted images were obtained. Based on these images, the applicant then advanced the infusion needle to the distance required to reach a point within the entorhinal cortex located 1 mm from the ventral brain surface (Figure 2).

[0130] Monkeys received 1 to 3 infusion sites into the entorhinal cortex on each side of the brain. For a single infusion on each side, the injection was placed approximately midway along the anterior-posterior length of the hippocampus. For the entorhinal cortex that received 2 to 3 infusions, an attempt was made to diffuse the vector into a larger volume of the entorhinal cortex. For this study, 34 entorhinal cortexes were inoculated with AAV2-BDNF (3×10 11 vg / ml) infusions: 18 received a single infusion site on each side of the brain, 9 received two infusions, and 7 received three infusions.

[0131] When the infusion needle is about to penetrate the brain, the infusion pump is turned on at a rate of 3 μU min to maintain positive pressure and prevent the needle from clogging as the needle is advanced through the brain toward the target. Once the final target is confirmed on MRI, the infusion rate is adjusted from 1 pl / min to 3 pl / min to achieve diffusion of the vector through the region of the entorhinal cortex located in the 1 mm thick MRI slice containing the infusion needle. The applicant did not infuse a preset volume of vector, but continued to infuse until the target area of ​​interest (about 5 mm of the rostral-caudal range of the entorhinal cortex) was covered with gadoteridol. Scans were continuously obtained through the infusion process, as previously described [Su et al., MotTher. 2010; San Sebastian et al., Mol Ther Methods Clin Dev. 2014. ]. The volume of vector infused at each site ranged from 15 to 160 μL. Vital signs were continuously monitored during the operation.

[0132] After the infusion was completed, the subjects were returned to the operating room to remove the skull-based stereotaxic frame. The animals then received intramuscular injections of NSAIDs (Meloxicam) and buprenorphine (Buprenex) as part of postoperative analgesia management on the day following the convection-enhanced delivery (CED) infusion. Once the animals were returned to their cages, they were evaluated by veterinary staff twice a day for 5 days. Detailed standardized forms were filled out for each animal, including assessments of surgical site integrity, edema, infection, balance, movement, posture, food intake, and fecal and urine output. No abnormalities or signs of discomfort were reported.

[0133] Treating cognitive impairment

[0134] In the applicant's study reported herein, the previously used method for delivering genes to the entorhinal / hippocampal circuit to treat cognitive impairment has been substantially modified. These modifications are based on: 1) primate studies of gene therapy vectors delivered to the entorhinal cortex and hippocampus, and 2) clinical experience of expanding the use of MR guidance, convection-enhanced delivery (CED) and gadoteridol co-infusion in 3 current clinical gene therapy trials. These considerations involve new knowledge, i.e., the relationship between the volume of the infusion and the distribution of the carrier depends on the presence of the precise needle position in the entorhinal cortex, variable carrier dispersion in the gray matter and white matter, the perivascular space (acting as a carrier sinking area during carrier infusion), the irregular anatomical structure of the entorhinal cortex, and the possibility of carrier backflow along the infusion pipeline. These factors require a significantly higher range of carrier volumes to effectively fill the targeted brain structures, the specific rate of carrier infusion, and the specific carrier dose (concentration).

[0135] [The volume of the human entorhinal cortex is 1500 mm, as determined 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 distribution volume (Vi / Vd) in the brain ranges from 1:1 to 1:3 in gray matter (a 1:1 ratio indicates the same infusion volume and distribution volume, while a 1:3 ratio indicates an infusion volume that is 3 times the volume of brain tissue). Therefore, without being bound by theory, the applicant calculates that a total vector volume of 500 μl to 1500 μl is required to fill the entorhinal cortex. The applicant aims to fill at least half of this volume, i.e., 250 μl to 750 μl need to be infused into each hemisphere of the brain. Applicants distributed this vector volume across 3 to 4 infusion sites and limited the maximum infusion volume per hemisphere to 750 μl. Similar to other intracranial gene therapy trials in the brain, adequate coverage will require a range of infusion volumes per site to most effectively fill the irregular structure of the entorhinal cortex. MRI imaging can be used to track vector distribution and assess vector distribution in real time to guide infusion volumes within the range.

[0136] In monkeys, the applicant used 3×10 11 The volume of 60 μl to 435 μl was infused at a concentration of 50% to 100% of the entorhinal cortex at a concentration of 1.5 vg / ml of the vector to achieve 50% to 100% filling of the entorhinal cortex as shown in Table 1 below. 3 )[5] is 1 / 6 the volume of the human entorhinal cortex. Applicants' preclinical infusion volumes from monkey to human are predicted to achieve 50% EC coverage, with corresponding human infusion volumes ranging from 360 μl to 2610 μl per hemisphere or alternatively 250 μl to 750 μl.

[0137] Table 1

[0138] Applicants' initially cited volumes for infusion into humans (up to 125 μl per site for a maximum of 375 μl per hemisphere) were overly conservative. Applicants' detailed review of new results from additional monkeys and expanding recent experience with vector delivery in humans has resulted in the following new guidelines: Larger volumes than those initially cited are required to achieve adequate filling of target structures in humans.

[0139] The revised parameters were based on studies of an additional 25 monkeys, which allowed the applicant to develop this specific expertise. The previous dose described in the literature was found to be ineffective (1×10e1), and 3×1011 The minimum dose of 1000 mg / ml is the lowest feasible starting dose. To the best of the applicant's knowledge, this information has not been previously published.

[0140] Up to 4 infusions are required per hemisphere to effectively treat the human brain. The correct application volume for humans includes a range of 360 μl to 2610 μl per hemisphere. The literature describes that the lower carrier doses (1×10e10 to 1×10e11) known to the applicant are ineffective, while the higher doses known to the applicant are too high and will cause toxicity (higher than 3×10e12). The literature also points out a method that requires infusion in the range of 15 μl / site to 125 μl / site or 15 μl / site to 130 μl / site, which means that the maximum volume to be infused at each site is 125 μl, and the maximum total amount per hemisphere is 375 μl.

[0141] Using previously published methods, survival of monkeys was extended to as long as 24 to 30 months, which identified issues with mistargeting and seizures. To the best of applicants' knowledge, this is unpublished basic information regarding achieving safe delivery of vectors. In order to safely deliver vectors, it is important to accomplish delivery within a region of the EC restricted to its most ventromedial aspect, excluding any vector infusion into the subiculum of the entorhinal cortex and parasubiculum of the entorhinal cortex, and also excluding direct infusion into the hippocampus. Animals that received infusions not within these parameters developed seizures.

[0142] Also based on these long-term, unpublished monkey studies, the applicants have determined that 3×10 11 vg / ml to 1×10 13 The vector dose range of 10 vg / ml was considered as the maximum safe and tolerable dose range.

[0143] Nagahra et al. (2018), Gene Therapy (2018), Vol. 25: pp. 104-114, teaches guidance on the use of MRI to reliably infuse AAV2-BDNF into this cerebellar region using gadoteridol infusion. To the best of applicant's knowledge, the prior art does not provide specific methods for targeting sub-portions of the entorhinal cortex (and then the ventromedial region) and avoiding the pre-subiculum, para-subiculum, subiculum, and hippocampus, and the literature does not address precise volume considerations and vector concentrations for succession. For example, the literature does not disclose specific details regarding: 1) effective vector concentrations for use in humans (e.g., to the best of applicant's knowledge, published reports are 3×10 11 vg / ml, but the human dose recommended by the applicant is about 3×10 11 vg / ml, 1×10 12 vg / ml up to 1×10 13vg / ml. To the best of applicant's knowledge, the higher doses studied by applicant in monkeys have not been reported or published), 2) effective infusion volumes for use in humans.

[0144] To the best of applicant's knowledge, the literature has not identified optimal infusion rate parameters for achieving vector diffusion within the target region of the brain. Vector infusion rates using a continuous infusion pump were determined in non-human primates to fall within a specific range of 1 μl / min to 15 μl / min. This infusion rate achieves adequate vector diffusion and coverage of the intended target. Lower rates fail to diffuse sufficiently within the target structure; higher rates leak from the intended target region, posing a safety risk.

[0145] The choice of design for delivering genes to the infusion needle in the brain should be based on testing of infusion needles specifically designed for different areas of the brain. In the entorhinal cortex, the applicant found that the "step" design of the needle is an effective way to prevent infusion backflow along the needle duct, in which the first few millimeters of the needle are narrow, followed by expansion of the diameter of the needle. In the entorhinal cortex region, the optimal needle design includes a single widened "step" (external diameter expansion) located at a distance of 1mm to 5mm from the tip of the infusion needle. This reduces vector backflow and losses on the infusion duct. Unintentional diffusion can compromise safety. This knowledge is essential for the effective implementation of gene therapy vectors in this brain region.

[0146] Carrier concentration: lower doses are ineffective, and higher doses are toxic: establish the ideal range

[0147] Select 3×10 11 The use of a minimum effective vector titer of vg / ml is based on an earlier primate study1006 (see Table 2), which found that 1 × 10 11 The concentration of vg / ml is not enough to effectively treat the targeted brain region, the entorhinal cortex. The rostral-caudal length of the human entorhinal cortex is 30 mm, but at 1×10 11 vg / ml titers drove BDNF expression only within 1 mm of the infusion site. Despite the use of convection-enhanced delivery (CED) and the infusion volume being similar to that of patients receiving 3 × 10 11 vg / ml, the infusion volume used was comparable, but this was the case. Therefore, the applicant concluded that 1×10 11 Titers of vg / ml are insufficient to treat a sufficient volume of the entorhinal cortex in humans with AD degeneration.

[0148] In primate study 1006 (see Table 2), the applicants also tested 3×10 11vg / ml of vector titer. Vector infusion at this concentration drove BDNF expression up to 8 mm from the infusion site (mean spread 5.21 ± 0.71 mm / site), using a similar titer as 1 × 10 11 vg / ml infusion volume. Primate studies 1006 (see Table 2) also found that 3×10 11 The vector concentration of 100 vg / ml was safe. Therefore, it was assumed that this was the lowest vector concentration that effectively delivered BDNF to a sufficient volume of the entorhinal cortex, and this was called the minimum effective vector concentration. Prior to this work, it was thought that lower vector doses might be effective. But based on the new and extensive primate distribution and dosing studies, they are not.

[0149] About 1×10 12 vg / ml to 1×10 13 Higher vector titers of 10 vg / ml: Primate studies 1004 (see Table 2) have shown that this dose is tolerated when precisely targeted to the entorhinal cortex. These titers are as high as 10 13 Injection of 100 vg / ml resulted in good entorhinal expression without the toxicity (seizures) associated with higher doses or vector mistargeting. The triangle below indicates the area of ​​BDNF gene expression after vector infusion. See Figure 7.

[0150] This study demonstrated that infusion of AAV2-BDNF into regions adjacent to the entorhinal cortex (hippocampus, amygdala, presubiculum, parasubiculum, and subiculum) in 5 monkeys resulted in safety issues (seizures). This toxicity was previously unknown and unexpected, and highlights the need for precise infusion into target regions of the entorhinal cortex to safely and effectively treat memory disorders.

[0151] New vector volume ranges are needed to accurately and safely treat memory disorders

[0152] The volume of the human entorhinal cortex is 1500 mm, as determined both by 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 distribution volume (Vi / Vd) in the brain ranges from 1:1 to 1:3 in gray matter (a 1:1 ratio indicates the same infusion volume and distribution volume, while a 1:3 ratio indicates that the infusion volume is 3 times the volume of brain tissue). Therefore, the applicant calculated that a total vector volume of 500 μl to 1500 μl is required to fill the entorhinal cortex. The applicant's goal in this experiment is to fill at least half of this volume, that is, 250 μl to 750 μl need to be infused into each hemisphere of the brain. Applicants propose to distribute this vector volume over 3 to 4 infusion sites, and propose to limit the maximum infusion volume per hemisphere to 750 μl. Similar to other intracranial gene therapy trials in the brain, adequate coverage will require a range of infusion volumes per site to most effectively fill the irregular structure of the entorhinal cortex. Furthermore, because Applicants use MR imaging to track vector distribution, Applicants will be able to accurately assess vector distribution in real time to guide infusion volumes within the range.

[0153] In monkeys, the applicant used 3×10 11 Volumes ranging from 60 μl to 435 μl were infused at vector concentrations of 50% to 100% fill of the entorhinal cortex, as shown in Table 1 above. Entorhinal cortex volume of rhesus monkeys (255 mm 3 ) [Paxinos et al., The Rhesus Monkey Brain in Stereotaxic Coordinates. (Academic Press, 1989)] is 1 / 6 the volume of the human entorhinal cortex. Applicants' preclinical infusion volumes from monkey to man were predicted to achieve 50% EC coverage, and the corresponding human infusion volumes would range from 250 μl to 2610 μl per hemisphere. These volumes are much higher than originally anticipated and published in the literature; the inventors have determined new parameters through extensive empirical testing in monkeys and more recent human experience. Consideration of the scaling factor from monkey to man has been critical in Applicants' clinical experience to date, where higher volumes than originally predicted have been required to achieve adequate filling of the target structures in humans. Infusion rates must be precisely controlled to accurately and safely treat memory disorders

[0154] Previous methods of delivering AAV into the parenchyma by simple injection were unable to effectively distribute the vector over large volumes of the target tissue. In contrast, a technique called CED (convection-enhanced delivery) uses a specially designed anti-reflux cannula to allow high infusion pressures to be applied at the cannula tip, thereby overcoming interstitial pressures in the brain and allowing the infusate to penetrate large volumes of the target area in a uniform manner. In addition, a chelated gadolinium contrast agent (gadoteridol) is included in the infusate in order to monitor the distribution of the infusate by magnetic resonance (MR) imaging. The distribution of gadoteridol closely matches that of the infused AAV2 vector, and there is no evidence of toxicity 7-9 Preclinical and clinical studies have shown that co-delivery of gadoteridol (a widely used MR imaging contrast agent that is marketed for intravenous administration and used off-label in this protocol) and real-time MR imaging helps ensure that the target area is exposed to the agent of study while minimizing exposure of non-target CNS tissues. 10,11 MR images obtained during the CED procedure will be analyzed post-procedure to measure the volume of gadoteridol contrast enhancement. The total gadoteridol distribution volume and the percentage of the entorhinal cortex covered by gadoteridol will be calculated. Correlations between coverage and other outcome measures, such as clinical cognitive measures, can be assessed.

[0155] In extensive preclinical testing on monkeys using infusion parameters ranging from 0.1 μl / min to 15 μl / min, the applicant has identified a new infusion rate range that supports consistent, safe and effective gene delivery to the entorhinal cortex for the treatment of memory disorders: 1 μl / min to 15 μl / min. Infusion rates below this value can result in insufficient MRI diffusion, while higher infusion rates can result in local tissue damage. To the best of the applicant's knowledge, this range is novel and inventive compared to the prior art, and this rate is critical to practicing the technology. See Table 2.

[0156] Table 2: Preclinical studies - Overview of AAV2-BDNF infused into the entorhinal cortex

[0157] An infusion cannula with a stepped design specifically adapted for the entorhinal cortex is needed to accurately and safely treat memory disorders

[0158] As mentioned above, a technique called CED (convection enhanced delivery) uses a specially designed anti-reflux cannula to allow high infusion pressure to be applied at the cannula tip, thereby overcoming the interstitial pressure in the brain and allowing the infusate to penetrate into a large volume of the target area in a uniform manner. The step design in the infusion catheter has been developed, with special consideration of the gray matter and white matter anatomy of the target area of ​​the entorhinal cortex. A variety of step designs have been tested, and the applicant has found that the optimal step distance is 0.5mm to 2.0mm from the infusion tip. This distance is necessary to prevent reflux into the subcortical white matter and loss of carriers in the wrong target. The step distance is not obvious and is an empirical test result in the entorhinal cortex of the rhesus monkey brain. As shown below, the previous step design has been at a longer distance from the sui target (such as the striatum) in the treatment of Parkinson's disease. This design applied to the entorhinal cortex will be ineffective.

[0159] Equivalent

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

[0161] The invention exemplarily described herein may be suitably implemented in the absence of any one or more elements, any one or more limitations not specifically disclosed herein. Thus, for example, the terms "comprises," "comprising," "containing," and the like are to be understood broadly and not restrictively. In addition, the terms and expressions employed herein have been used as descriptive terms rather than limiting terms, and when such terms and expressions are used, there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications are possible within the scope of the invention claimed.

[0162] Therefore, it should be understood that the materials, methods, and examples provided herein are representative of preferred embodiments, are exemplary only, and are not intended as limitations on the scope of the present 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 proviso or negative limitation deleting any subject matter from the genus, regardless of whether the deleted material is specifically recited herein.

[0164] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

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

[0166] Other embodiments are set forth in the following claims. References: 1.Kfomy N,Holmes BB,Jiang H,Holtzman DM,Diamond MI.Trans-cellularpropagation of Tau aggregation by fibrillar species.J Biol.Chem.2012;287:19440–51, 2. Mirbaha H, Holmes BB, Sanders DW, Bieschke I, Diamond MI. Tau trimers are the minimal propagation unit spontaneously internalized to seedintracellular aggregation. J Biol Chem. 2015; 290:14893–903. 3. Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 1991; 82: 239–59. 4. Su JH, Deng G, Cotman CW. Transneuronal degeneration in the spread of Alzheimer's disease pathology: immunohistochemical evidence for the transmission of tau hyperphosphorylation. Neu-robiol Dis. 1997; 4:365–75. 5.de Calignon A,Polydoro M,Suarez-Calvet M,William C,Ada-mowicz DH,Kopeikina Kr,et al.Propagation of tau pathology in a model of earlyAlzheimer’s disease.Neuron.2012;73:685–97. 6.Gomez-Isla T,Price JL,McKeel DW,Moths JC,Growdon JH,HymanBT.Profound loss of layer II entorhinal cortex neurons occurs in very mildAlzheimer’s disease.J Neurosci.1996;16:4491–4500. 7.Kordower JH,Chu Y,Stebbins GT,DeKosky ST,Cochran EJ,Bennett D,etal.Loss and atrophy of layer II entorhinal cortex neurons in elderly peoplewith mild cognitive impairment.Ann Neurol.2001;49:202–13. 8.Nagahara AH,Tuszynski MH.Potential therapeutic uses of BDNF inneurological and psychiatric disorders.Nat Rev Drug Disoov.2011;10:209–19. 9.Connor B,Young D,Yan Q,Faull RL,Synek B,Dragunow M.Brain-derivedneurotrophic factor is reduced in Alzheimer’s disease.Brain Res Mol BrainRes.1997;49:71–81. 10.Narisawa-Saito M,Wakabayashi K,Tsuji S,Takahashi H,Nawa H.Regionalspecificity of alterations in NGF,BDNF and NT-3 levels in Alzheimer's disease.Neuroreport.1996:7:2925–8. 11.Nagahara AH,Mateling M,Kovacs I,Wang L.,Eggert S,Rock-enstein E,etal.Early BDNF treatment ameliorates cell loss in the entorhinal cortex of APPtran sgen ic mice.J Neurosci.2013:33:15596–602. 12.Nagahara AH,Merrill DA,Coppola G,Tsukada 5,Schroeder BE,Shaked GM,et al.Neuroprotective effects of brain-derived neu-rotrophic factor in rodentand primate models of Alzheimer's disease.Nat Med.2009:15:331–7. 13.Poduslo IF,Curran GL.Permeability at the blood-brain and blood-nerve barriers of neurotrophic factors: NGF, CNTF, NT-3, BDNF.Brain Res MotBrain Res.1996:36:280–6. 14.Tuszynski MH,Yang JH,Beard D,U HS,Bakay RA,Pay MM,et al.Nerve growth factor gene therapy: activation of neuronal responses in Alzheimer's disease.JAMA Neurol.2015:72:1139–47. 15.Mai JK,Assheuer J,Paxinos G.Atlas of the Human Brain,AcademicPress:San Diego,2004. 16.Juottonen K,l Imkso MP,Insausti R,Lehtovirta M,Pitkanen A,PartanenK,et al.Volumes of the entorhinal and perirhinal cortices in Alzheimer’sdisease.Neurobiol Aging.1998;19:15–22. 17.Pennanen C,Kivipelto M,Tuornainen 5,Hartikainen P,Hanninen T,Laakso MP,et al.Hippocampus and entorhinal cortex in mild cognitiveimpairment and early AD.Neurobiol.Aging.2004;25:303–10. 18.Saito R,Bringas JR,McKnight TR,Wendland MF,Mamot C,Drummond DC,etal.Distribution of liposomes into brain and rat brain tumor models byconvection-enhanced delivery monitored with magnetic resonance imaging.CancerRes.2004;64:2572-9. 19.Krauze MT,McKnight TR,Yamashita Y,Bringas J,Noble CO,Saito R,etal.Real-time visualization and characterization of liposomal delivery intothe monkey brain by magnetic resonance imaging.Brain Res Brain ResProtoc.2005;16:20–6. 20.Fiandaca MS,Varenika V,Eberling J,McKnight T,Bringas J,PivirettoP.et al.Real-time MR imaging of adeno-associated viral vector delivery to theprimate brain.Neuroimage.2009;47 Suppl 2:T27–35. 21.Su X,Kells AP,Aguilar Salegio EA,Richardson RM,Hadaczek P,Beyer J,et al.Real-time MR imaging with Gadoteridol predicts distribution oftransgenes after convection-enhanced delivery of AAV2 vectors.Mot Ther.2010;18:1490–5. 22.Krauze MT,Forsayeth J,Yin D,Bankiewicz KS.Convection-enhanceddelivery of liposomes to primate brain.Methods Enzymol.2009;465:349–62. 23.Jahangiri A,Chin A,Flanigan PM,Chen R,Bankiewicz K,AghiMK.Convection enhanced delivery in glioblastoma a review of pectinical andclinical studies.J Neurosurg.2017;126:191-200.In press 24.Richardson RM,Kells AP,Rosenbluth KH,Salegio EA,Fiandaca MS,LarsonPS,et al.Interventional MRI-guided putaminal delivery of AAV2-GDNF for aplanned clinical trial in Parkinson’s disease.Mol Ther.2011;19:1048–57. 25.San Sebastian W,Kells AP,Bringas J,Samaranch L,Hadaczek P,Ciesielska A,et al.Safety arid tolerability of MRI-guided infusion of AAV2-hAADC into the mid-brain of non-human primate.Mol Ther Methods Clin Dev.2014;3:14049. 26.Richardson RM,Kells AP,Martin AJ,Larson PS,Stair PA,Piferi PG,etal.Novel platform for MRI-guided convection-enhanced delivery oftherapeutics:preclinical validation in nonhuman primate brain.Stereotact FundNeurosurg.39.2011;89:141–51. 27.Insausti R,Amaral DG,Cowan WM.The entorhinal cortex of the monkey:II.Cortical afferents.J Comp Neurol.1987;264:356–95. 28.Witter MP,Van Hoesen GW,Amaral DG.Topographical orga-nization ofthe entorhinal projection to the dentate gyms of the monkey.I Neurosci.1989;9:216–28. 29.Witter MP,Amaral DG.Entorhinal cortex of the monkey:V.Projectionsto the dentate gyms,hippo-campus,and subicular complex.J Comp Neurol.1991;307:437–59. 30.Amaral DG.Emerging principles of intrinsic hippo-campalorganization.Curr Opin Neurobiol.1993;3125–9. 31.Lavenex P,Amaral DG.Hippocampal-neocortical interaction:ahierarchy of associativity.Hippocampus.2000;10:420–30. 32.Kirkby DL,Higgins GA.Characterization of perforant path lesions inrodent models of memory and attention.Eur J Neurosci.1998;10:823–38. 33.Myhrer T,Naevdal GA.The temporal-hippocampal region and retention:the role of temporo-entorbinal connections in rats.Scand J Psycho’.1989;30:72–80. 34.Paxinos G,Huang XF,Toga AW.The Rhesus monkey brain in stereotaxiccoordinates.Academic Press;San Diego,1989. 35.Salegio EA,Kells AP,Richardson RM,Hadaczek P,Forsayeth 1,BringasJ,et al.Magnetic resonance imaging-guided delivery of adeno-associated virustype 2 to the primate brain for the treatment of ly sosomal storagedisorders.Hum Gene Ther.2010;21:1093-103. 36.Gimenez F,Krauze MT,Valles F,Hadaczek P,Bringas J,Sharma N.etal.Image-guided convection-enhanced delivery of GDNF protein into monkeyputamen.Neuroimage.2011;54 Suppl 1:5 1 89–95. 37.Liu L,Drouet V,Wu JW,Witter MP,Small SA,Clelland C,et al.Trans-synaptic spread of tau pathology in vivo.PLoS ONE.2012;7:El 1302. 38.Takeda S,Wegmann 5,Cho H,DeVos SL,Commins C,Roe AD,et al.Neuronaluptake and propagation of a rare phosphorylated high-molecular-weight tauderived from Atzheimer’s disease brain.Nat Commun.2015;6:8490. 39.Olsson B,Lautner R,Andreasson U,Ohrfelt A,Portelius E,Bjerke M,etal.CSF and blood biomarkers for the diagnosis of Alzheimer’s disease:asystematic review and meta-analysis.Lancet Neurol.2016;15:673–84. 40.Dubois B,Hampel H,Feldman HH,Scheltens P,Aisen P,Andrieu 5,etal.Preclinical Alzheimer’s disease:definition,natural history,and diagnosticcriteria.ALzheimers Dement.2016;1 / 292–323. 41.Bevan AK,Duque 5,Foust KD,Morales PR,Braun L,Schmetzer L,etal.Systemic gene delivery in large species for targeting spinal cord,twain,and peripheral tissues for pediatric disorders.Mol Ther.2011;19:1971–80. 42.Arvanitakis Z,Tuszyn ski MH,Bakay R,Arends D,Potkin 5,Bartus R,etal.Interim data from a phase 1 clinical trial of AAV-NGF(CERE-110)genedelivery in Atzheimers disease.AbstrAm Acad Neurol.2007:p05.071. 43.Rafii MS,Baumann TL,Bakay RA,Ostrove JM,Siffert J,Fleisher AS.etal.A phase’.study of stereotactic gene delivery of AAV2-NGF for Alzheimer’sdisease.Atzheimers Demerit.2014;10:571–81. 44.Fiandaca MS,Forsayeth JR,Dickinson PJ,Bankiewicz KS.Image-guidedconvection-enhanced delivery platform in the treatment of neurologicaldiseases.Neurotherageutics2008;5(1):123–7. 45.Salegio EA,Bringas J,Bankiewicz KS.MRI-guided delivery of viralvectors.Methods Mol Biol.2016;1382:217–30. 46.Amaral DG,Insausti R,Cowan WM.The entorhinal cortex of the monkey:I.Cytoarchitectonic organization.J Comp Neural.1987;264:326–55.

Claims

1. A method for improving cognitive function in a subject in need thereof, the method comprising administering about 3×10 11 vg / ml to about 1×10 13 vg / ml of a polynucleotide encoding brain-derived neurotrophic factor (BDNF) is administered to the ventromedial nucleus of the subject 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, thereby improving the cognitive function of the subject.

2. The method of claim 1, wherein the subject suffers from a condition selected from Alzheimer's disease (AD), mild cognitive impairment, presymptomatic AD, frontotemporal dementia, or dementia with Lewy bodies.

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

4. The method according to any one of claims 1 to 3, wherein the polynucleotide further comprises an expression vector, and the polynucleotide is operably linked to a regulatory nucleotide to drive expression of the polynucleotide.

5. The method according to any one of claims 1 to 4, wherein the administration is performed by convection enhanced delivery (CED).

6. The method of any one of claims 1 to 5, wherein the CED comprises an infusion catheter having a step distance of about 0.5 mm to about 2.0 mm from an infusion tip.

7. The method of any one of claims 1 to 6, wherein administration is not to the presubiculum, parasubiculum, subiculum or hippocampus.

8. The method according to any one of claims 1 to 7, wherein the polynucleotide is administered at 3 or 4 infusion sites.

9. The method according to any one of claims 1 to 8, wherein the dose comprises at least 3×10 11 vg / ml.

10. The method according to any one of claims 1 to 9, wherein the administration comprises a dose selected from the group consisting of 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 is about 7×10 11 vg / ml, 6×10 11 vg / ml is about 8×10 11 vg / ml, about 7×10 11 vg / ml is 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 is about 2×10 12 vg / ml, about 1×10 12 vg / ml to about 3×10 12 vg / ml, 2×10 12 vg / ml is 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 is about 7×10 12 vg / ml, 6×10 12 vg / ml is about 8×10 12 vg / ml, 7×10 12 vg / ml is about 9×10 12 vg / ml, or 8×10 12 vg / ml to about 1×10 13 vg / ml.

11. The method according to any one of claims 1 to 10, wherein the expression vector is selected from a plasmid, a liposome, a lentiviral vector, an adenoviral vector or an adeno-associated vector (AAV).

12. A method for delivering an expression vector to the ventromedial nucleus of a subject in need thereof, the method comprising infusing the vector by: (a) an infusion catheter having a step distance from the infusion tip of about 0.5 mm to about 2.0 mm; (b) an infusion rate of about 0.001 ml / min to about 0.015 ml / min; (c) an infusion volume of about 250 μl to about 750 μl per hemisphere, wherein the infusion occurs between about 3 to about 4 infusion sites; and (d) About 3×10 11 vg / ml to about 1×10 13 vg / ml dose, and Wherein the delivery avoids one or more of the following: the presubiculum, parasubiculum, subiculum, or hippocampus regions.

13. The method of claim 12, wherein the subject suffers from cognitive impairment.

14. The method of claim 12 or 13, wherein the expression vector further comprises a therapeutic polynucleotide.

15. The method of claim 14, wherein the polynucleotide encodes brain-derived neurotrophic factor (BDNF).

16. The method of any one of claims 12 to 15, wherein the administration comprises a dose selected from the group consisting of: 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 is about 7×10 11 vg / ml, 6×10 11 vg / ml is about 8×10 11 vg / ml, about 7×10 11 vg / ml is 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 is about 2×10 12 vg / ml, about 1×10 12 vg / ml to about 3×10 12 vg / ml, 2×10 12 vg / ml is 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 is about 7×10 12 vg / ml, 6×10 12 vg / ml is about 8×10 12 vg / ml, 7×10 12 vg / ml is about 9×10 12 vg / ml, or 8×10 12 vg / ml to about 1×10 13 vg / ml.

Images