Methods and vectors for treating CNS disorders

Abstract

A method for delivering a therapeutic protein to the central nervous system of a mammal is provided. [Solution] The method includes a step of administering rAAV particles containing AAV capsid proteins and a vector containing a nucleic acid encoding the therapeutic protein inserted between a pair of AAV inverted terminal repeat sequences to a non-central nervous system (CNS) cell, organ, or tissue of the mammal, the step being performed in a manner effective to infect the non-CNS cell, organ, or tissue within the mammal so that the non-CNS cell, organ, or tissue expresses and secretes the therapeutic protein into the circulation of the mammal, where the therapeutic protein is delivered to the central nervous system of the mammal.

Description

[Background technology]

[0001]

[0002] Treating central nervous system (CNS) diseases, such as Alzheimer's disease and other genetic brain disorders, remains a challenging problem. A key issue in treating brain diseases is that therapeutic proteins, when delivered intravenously, do not cross the blood-brain barrier, or when delivered directly to the brain, they do not distribute widely. Therefore, therapies for treating Alzheimer's disease need to be developed.

[0002]

[0003] Several different human apolipoprotein E (ApoE) isoforms exist, and the presence of some of these isoforms in the brain increases the risk of Alzheimer's disease (AD), while the presence of others decreases the risk. The presence of the ApoEε4 isoform is a strong genetic risk factor for late-onset sporadic AD (Casellano et al., Sci Transl Med, 3(89):89ra57 (29 June 2011)). The ApoEε4 allele significantly increases the risk of AD and lowers the age of onset. On the other hand, the presence of the ApoEε2 allele appears to decrease the risk of AD. Human ApoE isoforms are suggested to differentially affect amyloid-beta (Aβ) efflux or synthesis in vivo. [Overview of the Initiative]

[0003]

[0004] In certain embodiments, the present invention provides a method for treating a mammalian disease, comprising the step of administering to mammalian non-central nervous system (CNS) cells, organs, or tissues for delivery to the mammalian CNS (e.g., the brain). In certain embodiments, the present invention provides a method for treating a mammalian disease, comprising the step of administering to mammalian non-ocular cells, organs, or tissues for delivery to mammalian ocular cells, organs, or tissues.

[0004]

[0005] Mammals can be administered a vector containing rAAV particles with AAV capsid protein and nucleic acid encoding a therapeutic protein, inserted between a pair of AAV terminal inverted repeat sequences, in a manner effective for infecting non-CNS cells, organs, or tissues.

[0005]

[0006] In certain embodiments, non-CNS cells, organs, or tissues, and non-ocular cells, organs, or tissues include mammalian endocrine cells, organs, or tissues. Representative endocrine cells, organs, or tissues include liver cells, organs, and tissues, as well as pancreatic cells, organs, and tissues. In certain embodiments, liver cells, organs, or tissues are hepatocytes or include them.

[0006]

[0007] In certain embodiments, the present invention provides a method for delivering a protective ApoE isoform to the CNS of a non-rodent mammal by delivering or administering it to non-CNS cells, organs, or tissues of the non-rodent mammal (e.g., not to cerebrospinal fluid (CSF) or the brain). In one embodiment, in an effective manner for infecting non-CNS cells within a non-rodent mammal so that the non-CNS cells secrete the protective ApoE isoform into the systemic circulation (vascular structures or blood vessels) of the mammal, rAAV particles contain an AAV capsid protein, and the vector contains a nucleic acid encoding the protective ApoE isoform, inserted between a pair of AAV terminal inverted repeat sequences (ITRs). The protective ApoE isoform in circulation crosses the blood-brain barrier and enters the CNS (e.g., cerebrospinal fluid (CSF) or the brain, e.g., brain parenchyma).

[0007]

[0008] In certain embodiments, the present invention provides a method for delivering TPP1 (tripeptidyl peptidase I), CLN3 (battenin), PPT1 (palmitoyl protein thioesterase I), CLN6 (neuronal ceroid lipofuscinosis protein 6), or CLN8 to non-CNS cells, organs, or tissues of non-rodental mammals (for example, not to cerebrospinal fluid (CSF) or the brain). In one embodiment, in an effective manner for infecting non-CNS cells in non-rodent mammals so that non-CNS cells secrete TPP1, CLN3, PPT1, CLN6, or CLN8 into the systemic circulation (vascular structures or blood vessels) of mammals, rAAV particles contain an AAV capsid protein, and the vector contains nucleic acids encoding TPP1, CLN3, PPT1, CLN6, or CLN8, inserted between a pair of AAV terminal inverted repeat sequences. In circulation, TPP1, CLN3, PPT1, CLN6, or CLN8 cross the blood-brain barrier and enter the CNS (e.g., cerebrospinal fluid (CSF) or brain, e.g., brain parenchyma).

[0008]

[0009] In certain embodiments, the present invention provides a method for delivering a therapeutic protein to non-ocular cells, tissues, or organs of non-rodent mammals by delivery or administration to these cells, tissues, or organs of non-rodent mammals. In one embodiment, in an effective manner for infecting non-ocular cells, tissues, or organs within a non-rodent mammal so that the non-ocular cells, tissues, or organs secrete the therapeutic protein into the systemic circulation (vascular structures or blood vessels) of the mammal, rAAV particles contain an AAV capsid protein, and the vector contains a nucleic acid encoding the therapeutic protein, inserted between a pair of AAV terminal inverted repeat sequences. The circulating therapeutic protein crosses the blood-brain barrier and enters the ocular cells, tissues, or organs.

[0009]

[0010] In certain embodiments, the present invention provides a method for transfecting mammalian non-CNS cells, organs, or tissues for delivery to the mammalian CNS (e.g., cerebrospinal fluid (CSF) or brain, e.g., brain parenchyma). In one embodiment, the method comprises the step of delivering or administering to a mammalian endocrine cell, tissue, or organ (e.g., liver and / or pancreas) a vector comprising rAAV particles containing an AAV capsid protein and nucleic acids encoding a protective ApoE isoform, inserted between a pair of AAV terminal inverted repeat sequences, the vector being delivered in a manner effective for infecting the endocrine cell, tissue, or organ (e.g., liver and / or pancreas) for expression of the protective ApoE isoform and subsequent delivery to the mammalian CNS (e.g., cerebrospinal fluid (CSF) or brain, e.g., brain parenchyma) via, for example, systemic circulation (vascular structures or blood vessels). In another embodiment, the method comprises the step of delivering or administering to the liver and / or pancreas of a mammal, a vector comprising rAAV particles containing an AAV capsid protein and a nucleic acid encoding a protective ApoE isoform, inserted between a pair of AAV terminal inverted repeat sequences, such that the step is carried out in a manner effective in infecting endocrine cells, tissues, or organs (e.g., the liver and / or pancreas) for the expression of the protective ApoE isoform and subsequent delivery to the mammalian CNS (e.g., cerebrospinal fluid (CSF) or brain, e.g., brain parenchyma) via, for example, the systemic circulation (vascular structures or blood vessels).

[0010]

[0011] As used herein, the term “protective ApoE isoform” means an ApoE isoform that reduces one or more symptoms or signs of Alzheimer’s disease (e.g., physical, physiological, biochemical, histological, behavioral). Protective ApoE isoforms also mean ApoE isoforms that can reduce the risk of Alzheimer’s disease by at least 5%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc. or more.

[0011]

[0012] In certain embodiments, the present invention provides a method for treating lysosomal storage disorders or disorders. In certain embodiments, the disease or disorder is a deficiency or defect in the expression or activity of TPP1 (tripeptidyl peptidase I), CLN3 (battenin), PPT1 (palmitoyl protein thioesterase I), CLN6 (neuronal ceroid lipofuscinosis protein 6), or CLN8. In certain embodiments, the disease is a neurodegenerative disease, such as neuronal ceroid lipofuscinosis (NCL), e.g., infantile NCL, late-onset infantile NCL, juvenile NCL (Batten disease), and adult NCL. Other lysosomal storage disorders treated according to the present invention affect other tissues / organs, such as mucopolysaccharidosis (MPS IV and MPS VII), and can be treated using a vector containing rAAV particles containing AAV capsid protein and nucleic acids encoding therapeutic proteins, based on the method described herein.

[0012]

[0013] In certain embodiments, the disease or disorder is a neurodegenerative disease, such as neuronal ceroid lipofuscinosis (NCL), such as infantile NCL, late-onset infantile NCL, juvenile NCL (Batten disease), and adult NCL (inserted between a pair of AAV terminal inverted repeat sequences in a manner effective for infecting the liver and / or pancreas for the expression of the therapeutic protein and subsequent delivery to the mammalian CNS, for example, via the systemic circulation (vascular structures or blood vessels)). In one embodiment, the method comprises the step of delivering or administering to the liver and / or pancreas of a mammal the rAAV particle containing an AAV capsid protein and a vector containing nucleic acids encoding TPP1, CLN3, PPT1, CLN6, or CLN8, inserted between a pair of AAV terminal inverted repeat sequences, such that the step is carried out in a manner effective in infecting the liver and / or pancreas for the expression of TPP1, CLN3, PPT1, CLN6, or CLN8 and subsequent delivery to the mammalian CNS (e.g., the brain) via, for example, systemic circulation (vascular structures or blood vessels).

[0013]

[0014] In certain embodiments, the mammal is a non-rodent mammal. In certain embodiments, the non-rodent mammal is a primate, horse, sheep, goat, pig, or dog. In certain embodiments, the mammal is a human. In certain embodiments, the primate is a human. In certain embodiments, the human is a newborn, infant, child, teenager, or young adult.

[0014]

[0015] In certain embodiments, a mammal (e.g., human) has a defect or disorder of the central nervous system (CNS) that is treatable by gene replacement or suppression therapy.

[0015]

[0016] In certain embodiments, the coded defensive ApoE isoform has at least about 70% or more identity (e.g., 70-80% or 80-90%) with the ApoEε2 of a mammal (e.g., a primate, e.g., a human). In certain embodiments, the coded defensive ApoE isoform has 90-100% identity with the ApoEε2 of a mammal (e.g., a primate, e.g., a human).

[0016]

[0017] In certain embodiments, the coded TPP1 has at least about 70% or more identity (e.g., 70-80% or 80-90%) with the TPP1 of a mammal (e.g., a primate, e.g., a human). In certain embodiments, the coded TPP1 has 90-100% identity with the TPP1 of a mammal (e.g., a primate, e.g., a human).

[0017]

[0018] In certain embodiments, the coded CLN3, PPT1, CLN6, or CLN8 has at least about 70% or more identity (e.g., 70-80% or 80-90%) with the CLN3, PPT1, CLN6, or CLN8 of a mammal (e.g., a primate, e.g., a human). In certain embodiments, the coded CLN3, PPT1, CLN6, or CLN8 has 90-100% identity with the CLN3, PPT1, CLN6, or CLN8 of a mammal (e.g., a primate, e.g., a human).

[0018]

[0019] In certain embodiments, the encoded galactosamine-6-sulfatase has at least about 70% or more identity (e.g., 70-80% or 80-90%) with mammalian (e.g., primate, e.g., human) galactosamine-6-sulfatase. In certain embodiments, the encoded galactosamine-6-sulfatase has 90-100% identity with mammalian (e.g., primate, e.g., human) galactosamine-6-sulfatase.

[0019]

[0020] In certain embodiments, the encoded β-glucuronidase has at least about 70% or more identity (e.g., 70-80% or 80-90%) with mammalian (e.g., primate, e.g., human) β-glucuronidase. In certain embodiments, the encoded β-glucuronidase has 90-100% identity with mammalian (e.g., primate, e.g., human) β-glucuronidase.

[0021] In certain embodiments, a codon-optimized nucleic acid variant encoding a therapeutic protein is employed in the vector. In certain embodiments, such a codon-optimized nucleic acid variant achieves an increase in the transcription and / or translation of the encoded therapeutic protein. Such a codon-optimized nucleic acid variant may show, for example, a 0.5 to 10-fold increase in expression compared to a non-codon-optimized nucleic acid encoding the therapeutic protein.

[0020]

[0022] In certain embodiments, nucleic acid variants with reduced cytosine-guanine dinucleotide (CpG) encoding the therapeutic protein are employed in the vector. Examples of such cytosine-guanine dinucleotide (CpG)-reduced nucleic acid variants include those that show, for example, a 0.5 to 10-fold increase in expression for a specific CpG-reduced nucleic acid compared to a non-CpG-reduced nucleic acid encoding the therapeutic protein.

[0021]

[0023] In one embodiment, a nucleic acid variant encoding a therapeutic protein has a reduced cytosine-guanine dinucleotide (CpG) content compared to a nucleic acid that does not have reduced CpGs encoding the protein. In a special embodiment, the nucleic acid variant has at least 10 fewer cytosine-guanine dinucleotides (CpGs) than the nucleic acid that does not have reduced CpGs encoding the therapeutic protein. In an additional special embodiment, the nucleic acid variant has 20 or fewer, 15 or fewer, 10 or fewer, or 5 or fewer CpGs. In a more special embodiment, the nucleic acid variant has a maximum of 4, 3, 2, or 1 CpG. In a further special embodiment, the nucleic acid variant encoding a therapeutic protein has no cytosine-guanine dinucleotides (CpGs).

[0022]

[0024] In certain embodiments, the rAAV vector includes ITRs and / or capsids based on, or having sequence identity to, those of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8. In certain embodiments, the rAAV vector includes variants having less than 100% sequence identity to the ITRs and / or capsids of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8. Variants include amino acid insertions, additions, substitutions, and deletions. In particular embodiments, variants are described in International Publication 2013 / 158879 (International Application PCT / US2013 / 037170), International Publication 2015 / 013313 (International Application PCT / US2014 / 047670), and U.S. Patent Application Publication 2013 / 0059732 (U.S. Patent Application 13 / 594,773 disclosing LK01, LK02, LK03, etc.).

[0023]

[0025] In certain embodiments, the rAAV vector comprises one or more ITRs and / or capsids (VP1, VP2, and / or VP3) that are identical to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8 in an amount of at least 70-80%, 80-90%, or 90-99%. In certain embodiments, the rAAV vector includes ITRs (or multiple ITRs) and / or capsids (or multiple ITRs) (VP1, VP2, and / or VP3) having 75% or more sequence identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, etc.) with respect to any of the ITRs and / or capsids of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8.

[0024]

[0026] In certain embodiments, the rAAV vector comprises one or more ITRs and / or capsids (VP1, VP2, and / or VP3) having 100% identity with respect to AAV2 capsid (VP1, VP2, and / or VP3), AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8.

[0025]

[0027] In certain embodiments, the rAAV vector includes an AAV serotype or AAV pseudotype that includes an AAV capsid serotype different from the ITR serotype. When the ITR and capsid serotypes are different, the pseudotype rAAV in which the AAV capsid serotype is different from the ITR serotype is one of the ITRs and / or capsids of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8. It may consist of ITRs (multiple) and / or capsids (multiple) (VP1, VP2, and / or VP3) having 75% or more sequence identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, etc.) for any of them.

[0026]

[0028] In a particular embodiment, the AAV vector comprises VP1, VP2, and / or VP3 capsid sequences having 100% identity to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8, as well as one or more ITRs derived from different serotypes, for example, This includes AAV2ITR (AAV2 / 1) having an AAV1 capsid, AAV6ITR (AAV6 / 1) having an AAV1 capsid, AAV2ITR (AAV2 / LK03) having an LK01 capsid, AAV2ITR (AAV2 / 4-1) having an AAV4-1 capsid, AAV6ITR (AAV6 / LK03) having an LK01 capsid, or AAV6ITR (AAV6 / 4-1) having an AAV4-1 capsid.

[0027]

[0029] rAAV vectors may include additional components or elements that act in cis or trans. In a particular embodiment, a vector such as an rAAV vector further includes an intron, an expression regulatory element, one or more ITRs (e.g., any or a combination thereof of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, and / or AAV-2i8 serotypes), a filler polynucleotide sequence, and / or a poly(A) signal. In a particular embodiment, the intron is located within or adjacent to the nucleic acid encoding the therapeutic protein, and / or the expression regulatory element is operably linked to the nucleic acid encoding the therapeutic protein, and / or an AAV ITR(s) is adjacent to the 5' or 3' end of the nucleic acid encoding the therapeutic protein, and / or a filler polynucleotide sequence is adjacent to the 5' or 3' end of the nucleic acid encoding the therapeutic protein.

[0028]

[0030] In particular embodiments, the expression regulatory element includes a constitutive or modulotable regulatory element, or a tissue-specific expression regulatory element or promoter. In particular embodiments, the expression regulatory element includes an enhancer. In particular embodiments, the expression regulatory element (e.g., promoter or enhancer) induces expression in liver cells, organs, or tissues, or pancreatic cells, organs, or tissues. In particular embodiments, the expression regulatory element (e.g., promoter or enhancer) includes an element that induces expression in the liver (e.g., a TTR promoter or mutant TTR promoter).

[0029]

[0031] In certain embodiments, the present invention provides rAAV particles containing a vector comprising nucleic acids encoding a protective ApoE isoform, inserted between a pair of AAV ITRs, which are used to transfect non-CNS cells, organs, or tissues in mammals to produce therapeutic outcomes in the CNS. In one embodiment, the use is intended for the treatment of Alzheimer's disease in mammals.

[0030]

[0032] In certain embodiments, the present invention provides rAAV particles containing a vector comprising a nucleic acid encoding TPP1, inserted between a pair of AAV ITRs, which are used to transfect non-CNS cells, organs, or tissues in mammals to produce therapeutic results in the CNS. In one embodiment, the use is intended for the treatment of neuronal ceroid lipofuscinosis in mammals.

[0031]

[0033] In certain embodiments, the present invention provides rAAV particles containing a vector comprising nucleic acids encoding CLN3, PPT1, CLN6, or CLN8, inserted between a pair of AAV ITRs, which are used to transfect non-CNS cells, organs, or tissues in mammals to produce therapeutic results in the CNS. In one embodiment, the use is intended for the treatment of Batten disease in mammals.

[0032]

[0034] In certain embodiments, the present invention provides rAAV particles containing a vector comprising a nucleic acid encoding galactosamine-6-sulfatase, inserted between a pair of AAV ITRs, which are used to transfect non-ocular cells, organs, or tissues in mammals to produce therapeutic results in ocular cells, tissues, or organs. In one embodiment, the use is intended for the treatment of MPS IV.

[0033]

[0035] In certain embodiments, the present invention provides rAAV particles containing a vector comprising a nucleic acid encoding β-glucuronidase, inserted between a pair of AAV ITRs, which are used to transfect non-ocular cells, organs, or tissues in mammals to produce therapeutic results in ocular cells, tissues, or organs. In one embodiment, the use is intended for the treatment of MPS VII.

[0034]

[0036] In certain embodiments, the rAAV vector provides approximately 1 × 10¹⁶ vector genomes per kilogram of mammal. 8 ~1 × 10 10 pieces, 1×10 10 ~1 × 1011 cells, 1×10 11 to 1×10 12 cells, 1×10 12 to 1×10 13 cells, or 1×10 13 to 1×10 14 cells (vg / kg), is provided, administered, delivered, or used at a dose within this range. In certain embodiments, the rAAV vector is 1×10 vector genomes per kilogram of body weight 12 cells (vg / kg), is administered or used at a dose lower than this. In certain embodiments, the rAAV vector is about 5×10 vector genomes per kilogram of body weight of a mammal 11 cells (vg / kg), is administered or used at this dose.

[0035]

[0037] In a more specific embodiment, the amount of the rAAV vector provided, administered, delivered, or used is at least 1×10 vector genomes (vg) per kilogram of body weight of the mammal to achieve the desired therapeutic effect 10 cells (vg / kg), or about 1×10 vg per kilogram of body weight of the mammal 10 to 1×10 11 cells, or between about 1×10 vg per kilogram of body weight of the mammal 11 to 1×10 12 cells (e.g., between about 1×10 11 to 2×10 11 vg / kg, or about 2×10 11 to 3×10 11 vg / kg, or about 3×10 11 to 4×10 11 vg / kg, or about 4×10 11 to 5×10 11 vg / kg, or about 5×10 11 to 6×10 11 vg / kg, or about 6×10 11 to 7×10 11 vg / kg, or about 7×10 11 to 8×10 11 vg / kg, or about 8×10 11 to 9×10 11 vg / kg, or about 9×10 11 to 1×10 12(vg / kg) or approximately 1 × 10⁻⁶ vg per 1 kg of mammalian body weight. 12 ~1 × 10 13 The amount is between individuals. An additional special amount is needed to achieve the desired therapeutic effect, approximately 5 × 10⁶ vector genomes (vg) per kilogram of mammalian body weight. 10 ~1 × 10 10 It may be in the range of (vg / kg), or vg approximately 1 × 10 per kg of mammalian body weight. 10 ~5×10 11 It may be within the range of one, or per 1 kg of mammalian body weight, vg approximately 5 × 10 11 ~1 × 10 12 It may be within the range of one, or vg approximately 1 × 10 per kg of mammalian body weight. 12 ~5×10 13 It may also be within the range of an individual.

[0036]

[0038] In certain embodiments, the methods and / or uses of the invention do not induce or generate a substantial immune response to the therapeutic protein and / or rAAV particles in mammals, as described herein. In certain embodiments, mammals do not generate a substantial humoral immune response to the therapeutic protein and / or rAAV particles. In certain embodiments, a substantial immune response (e.g., humoral) to the therapeutic protein and / or rAAV particles is not generated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days, weeks, or months.

[0037]

[0039] In the context of treatment, a substantial immune response is considered to be a response that significantly reduces efficacy. Therefore, in the case of CNS disorders or diseases, if no detectable efficacy is observed when treating the CNS disorder or disease, this constitutes a substantial immune response. Consequently, a substantial immune response in the context of treatment does not mean a minimal immune response or an immune response that does not cause loss or significant reduction in efficacy.

[0038]

[0040] In certain embodiments, mammals do not develop a detectable immune response to the therapeutic protein and / or rAAV particles. In certain embodiments, mammals do not develop a detectable humoral immune response to the therapeutic protein and / or rAAV particles.

[0039]

[0041] In certain embodiments, mammals do not develop an immune response (e.g., humoral) to the therapeutic protein and / or rAAV particles that is sufficient to block the therapeutic effect of the therapeutic protein. In certain embodiments, mammals do not produce an immune response (e.g., humoral) to the therapeutic protein and / or rAAV particles that is sufficient to block the therapeutic effect for at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, or fourteen consecutive days, weeks, or months.

[0040]

[0042] In certain embodiments, empty capsids may be included in the rAAV vector, method, and use. If desired, empty AAV capsids can be added to the rAAV vector preparation or administered separately to a subject according to the method and use herein.

[0041]

[0043] In certain embodiments, empty AAV capsids are formulated together with rAAV vectors and / or administered to mammals. In special embodiments, empty AAV capsids are formulated in an amount equal to or less than the amount of vector (e.g., about 1.0 to 100 times the amount of rAAV vector relative to empty AAV capsids, or a ratio of about 1:1 of rAAV vectors to empty AAV capsids). In other special embodiments, rAAV vectors are formulated together with an excess of empty AAV capsids (e.g., more than 1 times the amount of empty AAV capsids relative to rAAV vectors, e.g., 1.0 to 100 times the amount of empty AAV capsids relative to rAAV vectors). Optionally, mammals with low to negative AAV NAb titers may receive smaller doses of empty capsid (1 to 10 times the amount of empty AAV capsid compared to the rAAV vector, 2 to 6 times the amount of empty AAV capsid compared to the rAAV vector, or approximately 4 to 5 times the amount of empty AAV capsid compared to the rAAV vector).

[0042]

[0044] In certain embodiments, the rAAV vector, method, and use include in the composition an excess of empty capsid in a dose or amount exceeding that of the rAAV vector (i.e., a vector containing nucleic acid encoding a therapeutic protein). The ratio of empty capsid to rAAV vector is approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5. 4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, or some other ratio.

[0043]

[0045] In some embodiments, the empty capsid contains the same proteins as the VP1, VP2, and VP3 capsid proteins present in the rAAV vector. In other embodiments, the empty capsid contains the VP1, VP2, and VP3 proteins having amino acid sequences different from those found in the rAAV vector. Optionally, if the capsid proteins of the empty capsid and the capsids of the rAAV vector are not identical in sequence, they are proteins of the same serotype.

[0044]

[0046] In certain embodiments, an empty capsid is included in the rAAV vector, method, and use, in which case the rAAV vector is a specific dose or amount. In some embodiments, the dose of the rAAV vector is approximately 1 × 10⁶ g per kg of mammalian body weight. 10 ~1 × 10 11Per individual, or per 1 kg of mammalian body weight, approximately 1 x 10⁻¹⁶ grams (vg) 11 ~1 × 10 12 pieces (for example, approximately 1 x 10) 11 ~2×10 11 vg / kg, or approximately 2 × 10⁻⁶ 11 ~3×10 11 vg / kg, or approximately 3 × 10⁻⁶ 11 ~4×10 11 vg / kg, or approximately 4 × 10 11 ~5×10 11 vg / kg, or approximately 5 × 10 11 ~6×10 11 vg / kg, or approximately 6 × 10⁻⁶ 11 ~7×10 11 vg / kg, or approximately 7 × 10⁻⁶ 11 ~8×10 11 vg / kg, or approximately 8 × 10 11 ~9×10 11 vg / kg, or approximately 9 × 10 11 ~1 × 10 12 Between vg / kg, or per 1kg of mammalian body weight, vg approximately 1 × 10 12 ~1 × 10 13 It is a capsid between individuals, and an empty capsid.

[0045]

[0047] In some embodiments, a predetermined dose or amount of rAAV vector, or a method or use utilizing a predetermined dose or amount of rAAV vector, optionally has an excess of empty capsid. In some embodiments, the dose of rAAV vector is approximately 1 × 10⁶ g per kg of mammalian body weight. 10 ~1 × 10 11 Per individual, or per 1 kg of mammalian body weight, approximately 1 x 10⁻¹⁶ grams (vg) 11 ~1 × 10 12 Between individuals (for example, approximately 1 × 10) 11 ~2×10 11 vg / kg, or approximately 2 × 10⁻⁶ 11 ~3×10 11 vg / kg, or approximately 3 × 10⁻⁶ 11 ~4×10 11 vg / kg, or approximately 4 × 10 11 ~5×10 11 vg / kg, or approximately 5 × 10 11 ~6×1011 vg / kg, or about 6×10 11 to 7×10 11 vg / kg, or about 7×10 11 to 8×10 11 vg / kg, or about 8×10 11 to 9×10 11 vg / kg, or about 9×10 11 to 1×10 12 vg / kg), or about 1×10 vg per kg of body weight of a mammal 12 to 1×10 13 particles, and the balance is excess empty capsids. Excess capsids relative to each dose or amount of rAAV vector may be about 1.5 to 100 times empty AAV capsids relative to rAAV vector. If desired, the ratio of empty capsids to rAAV vector relative to 1 can be about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.

[0046]

[0048] In certain embodiments, administration or delivery is by infusion or injection into the systemic circulation of the target. In certain embodiments, administration or delivery is by intravenous or intra-arterial infusion or injection into the systemic circulation of the target. In certain embodiments, administration or delivery is by infusion or injection into the hepatic portal vein of the target. In certain embodiments, administration or delivery is by implant or pump that enables infusion or injection into the systemic circulation of the target, or enables intravenous or intra-arterial infusion or injection into the systemic circulation of the target, or enables infusion or injection into the hepatic portal vein of the target. [Modes for carrying out the invention]

[0047]

[0049] The present invention is at least in part based on the development of an rAAV vector that, when administered to non-central nervous system (CNS) or non-ocular cells, tissues, or organs, can infect non-central nervous system (CNS) or non-ocular target cells, tissues, or organs for expression purposes, and subsequently enables the delivery of heterologous nucleic acid-encoded proteins to mammalian CNS or ocular cells, tissues, or organs. When an encoded protein is expressed by non-central nervous system (CNS) or non-ocular cells, tissues, or organs, the encoded protein is secreted into the systemic circulation and then delivered to mammalian CNS and / or ocular cells, tissues, or organs. Accordingly, the present invention provides a method for delivering proteins to mammalian CNS and / or ocular cells, tissues, or organs without direct administration to the mammalian CNS and / or ocular cells, tissues, or organs, i.e., by direct administration to cells, tissues, or organs other than mammalian CNS or ocular cells, tissues, or organs. Examples of such target cells, tissues, and organs include endocrine cells, tissues, and organs. A specific, non-limiting example is the liver (e.g., hepatocytes).

[0048]

[0050] Adeno-associated viruses (AAVs) are small, non-pathogenic viruses belonging to the parvoviridae family. AAVs are distinguished from other members of this family by their helper virus-dependent replication. In the absence of a helper virus, AAVs can integrate with the long arm (q) of chromosome 19 in a locus-specific manner. The approximately 5kb genome of AAV consists of a single segment of single-stranded DNA with either positive or negative polarity. The ends of the genome are folded into hairpin structures and contain short terminal inverted repeat sequences that can function as origins for viral DNA replication. Physically, parvovirus virions are unenveloped, and their icosahedral capsids are approximately 20–30 nm in diameter.

[0049]

[0051] As those skilled in the art will understand, AAV virions consist of three related proteins called VP1 proteins, two shorter proteins called VP2 proteins, and VP3, which is substantially an amino-terminal cleavage of VP1. Depending on the capsid and other factors known to those skilled in the art, the three capsid proteins VP1, VP2, and VP3 are generally present within the capsid in a ratio of approximately 1:1:10, respectively; however, this ratio, particularly the ratio of VP3, can vary significantly and should not be considered limiting in any respect.

[0050]

[0052] The terminal regions of the AAV genome contain short terminal inverted repeat sequences (ITRs) that can fold into a T-shaped hairpin structure that functions as the starting point for viral DNA replication. Within the ITR region, two elements central to ITR function, the GAGC repeat motif and terminal dissociation sites (trs), have been described. The repeat motif has been shown to bind to Rep when the ITR adopts a linear or hairpin conformation. This binding works to position Rep68 / 78 for cleavage at trrs that occurs in a site- and strand-specific manner. In addition to their roles in replication, these two elements appear to be central to viral integration. Among those contained within the chromosome 19 integration locus is a Rep-binding site containing adjacent trrs. Such elements have been shown to be functional for locus-specific integration.

[0051]

[0053] AAV is useful as a gene therapy vector because it can penetrate cells and introduce nucleic acids / genetic material so that the nucleic acids / genetic material can be stably maintained within the cell. Furthermore, such viruses can introduce nucleic acids / genetic material to specific sites, such as specific sites on chromosome 19. Since AAV is unrelated to pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial AAV pathogenesis or disease.

[0052]

[0054] Therefore, rAAV vectors, including serotypes and variants, provide means for delivering nucleic acid sequences into cells ex vivo, in vitro, and in vivo, where the nucleic acid sequence can encode a protein, and the encoded protein becomes expressible by the cell. For example, a recombinant AAV vector may contain heterologous nucleic acid encoding a desired protein or peptide (e.g., a protective apoE isoform). Delivery or administration of the vector to a subject (e.g., a mammal) thus provides the encoded protein to the subject.

[0053]

[0055] As used herein, the term “recombinant” as a modifier for AAV vectors, and for sequences such as recombinant nucleic acids and polypeptides, means that the composition (e.g., AAV or sequence) has been manipulated in a manner not commonly occurring in nature (i.e., engineered). A specific example of a recombinant AAV vector is the insertion of a nucleic acid genome that is not normally present in wild-type viruses (e.g., AAV) into the viral genome. A “recombinant” AAV vector is distinguished from the AAV genome because all or part of the viral genome is replaced with a non-natural sequence, such as a heterologous nucleic acid sequence, with respect to the AAV genome nucleic acid. The term “recombinant” is not necessarily used herein in relation to AAV vectors, and sequences such as nucleic acids and polypeptides, but recombinant forms of AAV, and sequences containing nucleic acids and polypeptides are clearly included regardless of any such omissions.

[0054]

[0056] Generally, in the case of AAV, one or both AAV terminal inversion repeat (ITR) sequences are retained within the AAV vector. The incorporation of non-native sequences (e.g., defensive apoE isoforms) thus defines the AAV vector as a "recombinant" AAV (rAAV) vector.

[0055]

[0057] Recombinant AAV vectors can be packaged – meaning “particles” for subsequent ex vivo, in vitro, or in vivo infection (transduction) of cells. When a recombinant AAV vector sequence is capsidized or packaged within an AAV particle, such particle may also be referred to herein as “rAAV”. Such particle contains a protein that capsidizes or packages the vector genome, in the case of AAV, a capsid protein.

[0056]

[0058] An "AAV virus particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein (generally all AAV capsid proteins) and a capsidized nucleic acid called a vector genome. If the particle contains heterologous nucleic acid, it is generally called "rAAV".

[0057]

[0059] The AAV vector “genome” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or capsidized to form AAV particles. When a recombinant plasmid is used to construct or manufacture a recombinant AAV vector, the AAV vector genome does not include any “plasmid” portion that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the “plasmid backbone” and is important for plasmid cloning and amplification, which are processes required for proliferation and recombinant AAV production, but it is not packaged or capsidized into rAAV particles itself.

[0058]

[0060] In a particular embodiment, the rAAV vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8, as well as capsids derived from their variants (e.g., capsid variants, e.g., insertion, addition, and substitution of amino acids). The serotype and variants of the rAAV vector include capsid variants (e.g., LK03, 4-1, etc.).

[0059]

[0061] rAAV serotypes and rAAV variants (e.g., capsid variants, e.g., LK03, 4-1, etc.) may or may not be different from other AAV serotypes (e.g., different from VP1, VP2, and / or VP3 sequences). As used herein, the term “serotype” is a characteristic used to refer to an AAV having a capsid that is serologically different from other AAV serotypes. Serological difference is determined by the lack of cross-reactivity between an antibody and one AAV compared to another AAV. Such differences in cross-reactivity usually result from differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Even if an AAV variant containing a capsid variant may not be serologically different from a reference AAV or other AAV serotype, the AAV variant will differ from a reference AAV or other AAV serotype in at least one nucleotide or amino acid residue.

[0060]

[0062] According to the conventional definition, a serotype means that the target virus has been tested for neutralizing activity against sera specific to all existing, characterized serotypes, but no antibody neutralizing the target virus has yet been found. As more natural virus isolates are discovered and / or capsid mutants are generated, they may or may not be serologically distinct from any of the currently existing serotypes. Therefore, if a new virus (e.g., AAV) does not have serological differences, this new virus (e.g., AAV) is a subgroup or a variant of the corresponding serotype. In many cases, serological testing for neutralizing activity is still performed on mutant viruses with altered capsid sequences to confirm whether they are viruses of a different serotype, based on the conventional definition of serotype. Therefore, for convenience and to avoid repetition, the term “serotype” broadly refers to both serologically distinct viruses (e.g., AAV) and serologically non-distinguishable viruses (e.g., AAV) that may be included in a subgroup or variant within a given serotype.

[0061]

[0063] Recombinant AAV vectors (e.g., rAAV), as well as methods and their use, include any viral strain or serotype. As a non-limiting example, a recombinant AAV vector genome may be based on any AAV genome, e.g., AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -rh74, -rh10, or AAV-2i8, etc. Such vectors may be based on the same lineage or serotype (or subgroup or variant), or they may be different from each other. As a non-limiting example, a recombinant AAV vector genome based on a single serotype genome may be identical to one or more of the capsid proteins packaging the vector. Furthermore, recombinant AAV vector genomes may be based on an AAV (e.g., AAV2) serotype genome different from one or more of the capsid proteins packaging the vector, in which case at least one of the three capsid proteins may be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, or a variant (e.g., a capsid variant, e.g., LK03, 4-1, etc.).

[0062]

[0064] AAV vectors therefore contain gene / protein sequences identical to those characteristic of a particular serotype. As used herein, “AAV vector associated with AAV1” means one or more AAV proteins (e.g., VP1, VP2, and / or VP3 sequences) that have substantial sequence identity to one or more polynucleotide or polypeptide sequences containing AAV1. Similarly, “AAV vector associated with AAV8” means one or more AAV proteins (e.g., VP1, VP2, and / or VP3 sequences) that have substantial sequence identity to one or more polynucleotide or polypeptide sequences containing AAV8. “AAV vector associated with AAV-Rh74” means one or more AAV proteins (e.g., VP1, VP2, and / or VP3 sequences) that have substantial sequence identity to one or more polynucleotide or polypeptide sequences containing AAV-Rh74 (see e.g., VP1, VP2, VP3). Such AAV vectors associated with other serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, are therefore derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, and AAV-2i8. While they may have a different number of sequences, they may exhibit substantial sequence identity to one or more genes and / or proteins from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, and / or possess one or more functional features (e.g., cell / tissue tropism). Capsid variant 4-1 of Example 6 is a representative and non-limiting example of AAV-Rh74 and related AAV variants.

[0063]

[0065] In various representative embodiments, the AAV vector associated with a reference serotype has a polynucleotide, polypeptide, or subsequence thereof that contains or is composed of a sequence that is at least 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8. Therefore, the methods and uses of the present invention include AAV sequences (polypeptides and nucleotides) and partial sequences thereof that exhibit 100% or less than 100% sequence identity with reference AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or AAV-2i8, for example, the AAV-Rh74 gene or protein sequence (for example, the VP1, VP2, and / or VP3 sequences described in Example 6), but may not be identical to known AAV genes or proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8.

[0064]

[0066] In one embodiment, an AAV polypeptide or a subsequence thereof includes or is composed of a sequence that is at least 70% or more identical, for example, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., to any of the reference AAV sequences or subsequences thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8, etc. (for example, the VP1, VP2, and / or VP3 sequences described in Example 6), i.e., up to 100% identical. In a particular embodiment, the AAV variant includes capsid variant 4-1 or LK03 VP1, VP2, and / or VP3 as described in Example 6. Recombinant AAV vectors, variants, hybrids, and chimeric sequences can be constructed using recombinant techniques known to those skilled in the art to include one or more heterologous nucleic acid sequences (transgenes) adjacent to one or more functional AAV ITR sequences. Such rAAV vectors may have, for example, one or more wild-type AAV genes lacking all or part of the rep and / or cap genes, but may retain at least one functional adjacent ITR sequence if necessary for the rescue, replication, and packaging of the recombinant vector into its AAV vector particles. The AAV vector genome thus contains sequences required in cis for replication and packaging (e.g., functional ITR sequences).

[0065]

[0067] "AAV ITR" or "AAV ITRs" refers to a region in the art recognized that is found at each end of the AAV genome and functions together in cis as a starting point for DNA replication and as a packaging signal for the virus. AAV ITRs, along with AAV rep coding regions, enable efficient excision and rescue of nucleotide sequences inserted between two adjacent ITRs, and their integration into the mammalian cell genome.

[0066]

[0068] The nucleotide sequences of AAV ITRs are publicly known. “AAV ITRs” do not need to have the indicated wild-type nucleotide sequence and may be altered, for example, by nucleotide insertions, deletions, or substitutions. Furthermore, AAV ITRs may originate from any of several AAV serotypes. Moreover, the 5' and 3' ITRs adjacent to heterologous nucleic acid sequences within an AAV vector do not necessarily need to be identical or derived from the same AAV serotype or isolate, as long as they function as intended, i.e., enabling the excision and rescue of the desired sequence derived from the host cell genome or vector, and enabling the integration of the heterologous sequence into the recipient cell genome.

[0067]

[0069] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to all forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short disruptive (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include natural, synthetic, and intentionally modified or altered polynucleotides (e.g., those with reduced CpG dinucleotides). Nucleic acids may be single-stranded, double-stranded, or triple-stranded, linear, or circular. When considering nucleic acids, the sequence or structure of a particular nucleic acid may be described herein according to the rule of presenting the sequence in the 5' to 3' direction.

[0068]

[0070] The rAAV vectors described herein contain exogenous (heterogeneous) nucleic acids functionally linked to a promoter. “Heterogeneous” nucleic acids mean nucleic acids inserted into an AAV vector for the purpose of transporting / delivering nucleic acids to cells, tissues, or organs via the vector. Heterogeneous nucleic acids are different from AAV nucleic acids, i.e., they are unnatural with respect to AAV nucleic acids. For example, in certain embodiments, the heterogeneous nucleic acid encodes a protective ApoE isoform.

[0069]

[0071] The term "heterogeneous" is not necessarily used in relation to nucleic acids in this specification; however, even in citations of nucleic acids where the modifier "heterogeneous" is not present, it is intended that such omissions imply the inclusion of heterogeneous nucleic acids. Once transported / delivered into cells, heterogeneous nucleic acids contained in rAAV vectors are expressible (e.g., transcribed and translated as appropriate).

[0070]

[0072] The term "transgene" is used herein to refer to, for convenience, a heterologous nucleic acid intended to be introduced into a cell or organism, or already introduced into a cell or organism. Examples of transgenes include any nucleic acid, such as a polypeptide or protein (e.g., a protective apoE isoform).

[0071]

[0073] In cells containing a transgene, the transgene is introduced / transferred by "infection" with an rAAV vector or by "transduction" of the cell. The terms "infection" and "transduction" refer to the introduction of molecules such as nucleic acids into a cell or host organism, for example, by an AAV vector. An "infected" or "transduced" cell (e.g., a cell, tissue, or organ cell in a mammal) refers to the genetic changes within the cell after the introduction of nucleic acid (e.g., a transgene). Therefore, an "infected" or "transduced" cell is, for example, a cell into which an exogenous molecule has been introduced or a replica thereof. The cell(s) are proliferative, the introduced nucleic acid is transcribable, and the protein is expressible. In the use and methods of gene therapy, transduced cells may be present in the target organism.

[0072]

[0074] Examples of cells that can be transduced include non-CNS cells, tissues, or organs. Examples of CNS cells, tissues, or organs include cerebrospinal fluid (CSF), the brain, intracranial spaces, and the spinal cord. Therefore, when referring to non-CNS cells, tissues, or organs, cerebrospinal fluid (CSF), the brain, intracranial spaces, and the spinal cord are excluded.

[0073]

[0075] Cells that can be transduced include non-ocular cells, tissues, or organs. Examples of ocular cells, tissues, and organs include the eye and parts of the eye. Therefore, when referring to non-ocular cells, tissues, or organs, the eye and parts of the eye are excluded.

[0074]

[0076] Non-CNS and non-ocular cell types include non-limiting examples such as liver cells (e.g., hepatocytes, sinusoidal endothelial cells) and pancreatic cells (e.g., β-islet cells). Other examples include skeletal muscle cells (e.g., fibroblasts).

[0075]

[0077] The polypeptides, proteins, and peptides encoded by nucleic acid sequences include full-length natural sequences such as natural proteins, as well as functional partial sequences, modified forms, or sequence variants, as long as they retain some degree of functionality of the natural full-length protein. In the methods and uses of the present invention, such polypeptides, proteins, and peptides encoded by nucleic acid sequences may, but do not have to be, identical to, a defective endogenous protein or an endogenous protein that is poorly expressed or deficient in the treated mammal.

[0076]

[0078] In one embodiment, a “therapeutic molecule” is a peptide or protein that may alleviate or reduce symptoms resulting from the absence or defect of a protein within a cell or subject. Alternatively, a “therapeutic” peptide or protein encoded by a transgene is a peptide or protein that provides a benefit to a subject, such as correcting a genetic defect or correcting a deficiency in a gene (expression or function).

[0077]

[0079] Non-limiting examples of heterogeneous nucleic acids encoding useful therapeutic proteins based on the present invention include heterogeneous nucleic acids that can be used in the treatment of diseases or disorders including, but not limited to, diseases or disorders of the CNS. Specific non-limiting examples include protective ApoE isoforms, e.g., sequences at least 70% identical to human ApoEε2; TPP1, e.g., sequences at least 70% identical to human TPP1; CLN3, e.g., sequences at least 70% identical to human CLN3; PPT1, e.g., sequences at least 70% identical to human PPT1; CLN6, e.g., sequences at least 70% identical to human CLN6; and CLN8, e.g., sequences at least 70% identical to human CLN8. Further specific non-limiting examples include galactosamine-6-sulfatase, e.g., sequences at least 70% identical to human galactosamine-6-sulfatase, and β-glucuronidase, e.g., sequences at least 70% identical to human β-glucuronidase.

[0078]

[0080] Non-exclusive examples of CNS diseases or disorders include Alzheimer's disease, lysosomal storage disorders, neuronal ceroid lipofuscinosis (NCL), such as infantile NCL, late-onset infantile NCL, juvenile NCL (Batten disease), adult NCL, etc., or mucopolysaccharidosis (e.g., MPS IV or MPS VII).

[0079]

[0081] The rAAV vectors described herein may optionally further include additional elements such as expression regulatory elements (e.g., promoters, enhancers), introns, ITRs(or ITRs), and polyadenine (also called polyadenylation) sequences. Generally, expression regulatory elements are sequences(or sequences) that affect the expression of operablely linked nucleic acids. Regulatory elements, including the expression regulatory elements described herein, such as promoters and enhancers present in the vector, are included to facilitate proper heterologous nucleic acid transcription and, where applicable, translation (e.g., splicing signals for promoters, enhancers, and introns, maintenance of the correct reading frame of genes to enable in-frame translation of mRNA, and stop codons). Such elements generally act in the cis and are called "cis-acting" elements, although they may also act in the trans.

[0080]

[0082] Expression control can be affected at levels such as transcription, translation, splicing, and message stability. Generally, regulatory elements that regulate transcription are juxtaposed near the 5' end of the nucleic acid being transcribed (i.e., "upstream"). Regulatory elements may also be located at the 3' end of the sequence being transcribed (i.e., "downstream") or within the transcript (e.g., within an intron). Regulatory elements can be located adjacent to the sequence being transcribed, or at a certain distance from it (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide), or even at a considerable distance. Nevertheless, due to length limitations of certain vectors, such as AAV vectors, such regulatory elements are generally located within 1-1000 nucleotides of the nucleic acid being transcribed.

[0081]

[0083] Functionally, an element (e.g., a promoter) can at least partially control the expression of a operably linked heteronucleotide so as to regulate the transcription of a polynucleotide and, if necessary, the translation of the transcript. A specific example of an expression regulatory element is a promoter, which is typically located at the 5' of the sequence being transcribed. Another example of an expression regulatory element is an enhancer, which may be located at the 5', 3', or within the sequence being transcribed.

[0082]

[0084] As used herein, "promoter" may mean a nucleic acid (e.g., DNA) sequence located adjacent to the polynucleotide sequence encoding the recombinant product. Promoters are typically operetically linked to adjacent sequences, such as heterologous nucleic acids. Promoters generally increase the expression level from heterologous polynucleotides compared to the expression level in the absence of the promoter.

[0083]

[0085] As used herein, "enhancer" may mean a sequence located adjacent to a heterologous polynucleotide. Enhancer elements are generally located upstream of promoter elements, but they also have function and may be located downstream of or within the DNA sequence (e.g., heterologous nucleic acid). Thus, enhancer elements may be 100 base pairs, 200 base pairs, or 300 or more base pairs, located upstream or downstream of heterologous nucleic acid. Enhancer elements generally increase the expression of heterologous nucleic acid more than the expression increase brought about by promoter elements.

[0084]

[0086] Examples of regulatory elements (e.g., promoters) include elements that are active in specific tissues or cell types, and are referred to herein as "tissue-specific regulatory elements / promoters." Tissue-specific regulatory elements are generally active in specific cells or tissues (e.g., liver, pancreas, muscle, etc.). Since regulatory elements are recognized by transcription activator proteins or other transcription regulators specific to certain cells, tissues, or organ types, regulatory elements are generally active in such cells, tissues, or organs.

[0085]

[0087] The promoter can be any desired promoter and may be selected based on known considerations, such as the expression level of the nucleic acid functionally linked to the promoter and the cell type in which the vector is used. The promoter can be exogenous or endogenous.

[0086]

[0088] Examples of active promoters in skeletal muscle include promoters derived from genes encoding skeletal muscle-type α-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, as well as synthetic muscle-type promoters that exhibit higher activity than native promoters (see, for example, Li et al., Nat. Biotech. 17:241-245 (1999)). Examples of liver-specific promoters include the human α1-anti-trypsin (hAAT) promoter; albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther. 3:1002-9 (1996); α-fetoprotein (AFP), Arbuthnot et al., Hum. Gene. Ther., 7:1503-14 (1996); bone (osteocalcin, Stein et al., Mol. Biol. Rep., 24:185-96 (1997); bone sialoprotein, Chen et al., J. Bone Miner. Res. 11:654-64 (1996)); and lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 Examples include immunoglobulin heavy chains (T cell receptor α chain) and TTR promoters (1998). Examples of active enhancers in the liver include apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0087]

[0089] Expression regulatory elements also include ubiquitous or mixed promoters / enhancers that have the ability to drive polynucleotide expression in many different cell types. Such elements include, but are not limited to, viral promoters, such as the cytomegalovirus (CMV) pre-early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in various mammalian cell types, or synthetic elements not found in nature (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the bovine papillomavirus promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerol kinase (PGK) promoter. Additional promoters include the inducible metallothionein promoter, AAV promoters (e.g., the AAVp5 promoter), promoters derived from actin genes, immunoglobulin genes, adenovirus promoters (e.g., the major late promoter of adenoviruses), the inducible heat shock promoter, and respiratory rash viruses.

[0088]

[0090] Expression regulatory elements can also induce expression in a regulated manner; that is, a signal or stimulus increases or decreases the expression of operably linked heterologous polynucleotides. Regulatory elements that increase the expression of operably linked polynucleotides in response to a signal or stimulus are also called “inducible elements” (i.e., they are triggered by a signal). A special example is, but is not limited to, hormone (e.g., steroid)-inducible promoters. Regulatory elements that decrease the expression of operably linked polynucleotides in response to a signal or stimulus are called “repressive elements” (i.e., the signal decreases expression so that expression increases when the signal is removed or absent). Generally, the increase or decrease brought about by such elements is proportional to the amount of signal or stimulus present; the greater the amount of signal or stimulus, the greater the increase or decrease in expression. Specific, non-limiting examples include: zinc-inducible sheep metallothionein (MT) promoter; steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; T7 polymerase promoter system (International Publication No. 98 / 10088); tetracycline-inhibiting system (Gossen et al., Proc. Natl. Acad. Sci.USA, 89:5547-5551 (1992)); tetracycline-inducible system (Gossen et al., Science. 268:1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); RU486-inducible system (Wang et al., Nat. Biotech. 15:239-243 (1997), and Wang et al., Gene Ther. 4:432-441) (1997)]; and rapamycin-inducible systems (Magari et al., J. Clin. Invest. 100:2865-2872 (1997); Rivera et al., Nat. Medicine. 2:1028-1032 (1996)). Other regulatory elements that may be useful in this context include those controlled by specific physiological conditions, such as temperature, acute phase, and development.

[0089]

[0091] As used herein, the terms “operable linkage” or “operable linkage” mean a physical or functional parallel arrangement of components that enables the components to function in the manner they are intended. In the example of an expression regulatory element operably linked to a nucleic acid, the regulatory element modulates the expression of the nucleic acid. More specifically, for example, two operable linkages of DNA sequences mean that the two DNA sequences are arranged (cis or trans) in a relationship where at least one of the DNA sequences can exert a physiological effect on the other sequence.

[0090]

[0092] Additional elements related to rAAV vectors and plasmids include, for example, filler or stuffer polynucleotide sequences that improve packaging and reduce the presence of contaminating nucleic acids, thereby reducing the packaging of the plasmid backbone. AAV vectors generally tolerate DNA insertions having a defined size range, typically about 4kb to about 5.2kb, or slightly more. Therefore, for shorter sequences, stuffers or fillers are incorporated into the insertion fragment to lengthen the viral genome sequence to near or exactly the normal size that allows for packaging of the AAV vector into the viral particle. In various embodiments, the filler / stuffer nucleic acid sequence is an untranslated (non-protein-coding) segment of the nucleic acid. In a particular embodiment of the AAV vector, the heterologous polynucleotide sequence has a length of less than 4.7 kb, and the filler or stuffer polynucleotide sequence, when combined with the heterologous polynucleotide sequence (e.g., inserted into the vector), has a total length of approximately 3.0 to 5.5 kb, or approximately 4.0 to 5.0 kb, or approximately 4.3 to 4.8 kb.

[0091]

[0093] As used herein, an AAV “empty capsid” does not contain the vector genome (hence the term “empty”), in contrast to a “genome-containing capsid” that contains the AAV vector genome. An empty capsid is a virus-like particle in that it reacts with one or more antibodies that react with the natural (genome-containing AAV vector) virus.

[0092]

[0094] While not intending to be constrained by theory, it is thought that empty AAV capsids function as decoys that bind to or react with antibodies against AAV vectors, thereby reducing the inactivation of the AAV vector. Such decoys work to absorb antibodies that target AAV vectors, thereby increasing or improving the transduction (transduction of the transgene) of the AAV vector into cells, and subsequently increasing the cellular expression of the transcript and / or encoded protein.

[0093]

[0095] Empty capsids can be manufactured and purified to the desired quality and quantity. For example, the titer of empty capsids can be measured by spectrophotometry from the optical concentration at a wavelength of 280 nm (based on Sommer et al., Mol. Ther. 2003 Jan;7(1):122-8).

[0094]

[0096] Empty AAVs or empty capsids are sometimes found naturally in AAV vector preparations. Such natural mixtures are usable under the present invention, or, if desired, can be manipulated to increase or decrease the amount of empty capsids and / or vectors. For example, the amount of empty capsids can be optionally adjusted to an amount expected to reduce the inhibitory effect of an antibody reacting with an AAV vector intended for use in vector-mediated gene transfer into a subject. The use of empty capsids is described in U.S. Patent Publication 2014 / 0336245.

[0095]

[0097] In various embodiments, empty AAV capsids are formulated together with rAAV vectors and / or administered to subjects. In a particular embodiment, empty AAV capsids are formulated in an amount equal to or less than that of the vector (e.g., about 1.0 to 100 times the amount of AAV vector relative to empty AAV capsids, or about 1:1 as the ratio of AAV vector to empty AAV capsids). In other particular embodiments, the AAV vectors are formulated together with an excess of empty AAV capsids (e.g., more than 1 times the amount of empty AAV capsids relative to the AAV vector, e.g., 1.0 to 100 times the amount of empty AAV capsids relative to the AAV vector).

[0096]

[0098] In some embodiments, the empty capsid contains the same VP1, VP2, and VP3 capsid proteins as those present in the rAAV vector. In other embodiments, the empty capsid contains the VP1, VP2, and VP3 proteins having different amino acid sequences than those found in the rAAV vector. Generally, but not always, if the capsid proteins of the empty capsid and the capsid of the rAAV vector are not identical in sequence, they are of the same serotype.

[0097]

[0099] Suitable mammals include humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domesticated animals (dogs and cats), livestock (poultry, e.g., chickens and ducks, horses, cattle, goats, sheep, pigs, etc.), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, young adults, and adults. Examples of animal disease models include mouse and other mammalian models known to those skilled in the art.

[0098]

[0100] Mammals suitable for treatment include animals that are at risk of or exposed to producing insufficient quantities, or animals that produce defective functional gene products (proteins) or abnormal, partially functional or non-functional gene products (proteins) that may cause disease. Subjects suitable for treatment according to the present invention also include subjects that are at risk of or exposed to producing abnormal or defective (mutant) gene products (proteins) that cause disease, such that reducing the quantity, expression, or function of the abnormal or defective (mutant) gene product (protein) would result in the treatment of the disease, suppression of one or more symptoms, or improvement of the disease.

[0099]

[0100] Thus, mammals may have a condition in which gene replacement therapy is effective. As used herein, “gene replacement therapy” means the administration of a protein-coding nucleic acid to a recipient and the subsequent in situ expression of the administered nucleic acid. Thus, the idiomatic phrase “a condition in which gene replacement therapy is effective” includes conditions such as genetic disorders (i.e., disease conditions resulting from one or more gene defects). According to one embodiment, the mammalian recipient has a genetic disorder, and the rAAV vector contains heterologous nucleic acid encoding a therapeutic protein for treating the disorder.

[0100]

[0101] The present invention provides a method for delivering nucleic acids to non-CNS cells, tissues, or organs, and a method for delivering nucleic acids to non-ocular cells, tissues, or organs, comprising the step of administering rAAV particles containing a vector comprising nucleic acids inserted between a pair of AAV terminal inverted repeat sequences to cells, tissues, or organs, thereby delivering the nucleic acids to the cells, tissues, or organs. The rAAV particles can remain in contact with cells for any desired time, and generally, the particles can remain indefinitely once administered. Provided that the CNS and / or ocular cells, tissues, or organs are not administered, administration to cells can be achieved by any means, including local, local, or systemic means.

[0101]

[0102] rAAV vectors may contain heterologous nucleic acids encoding protective ApoE isoform proteins. rAAV vectors infect non-CNS and / or non-ocular cells, leading to the expression and secretion of protective ApoE isoform proteins. The expressed and secreted protective ApoE isoform proteins enter the circulation and then enter the CNS.

[0102]

[0103] The rAAV expression vector can be constructed using known techniques and comprises a regulatory element containing at least a transcription start region, the target DNA, and a transcription end region, as an operationally linked component in the transcription direction. The regulatory element is selected to be functional in mammalian cells. The resulting construct contains the operationally linked component and is adjacent to a functional AAV ITR sequence (5' and 3').

[0103]

[0104] To produce rAAV virions, an AAV expression vector is introduced into suitable host cells using known techniques, such as transfection. Several transfection techniques are generally known in the art. See, for example, Sambrook et al., (1989) Molecular Cloning, laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transfection methods include co-precipitation with calcium phosphate, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-speed micro-injection.

[0104]

[0105] The "AAV rep coding region" refers to the region of the AAV genome that encodes the replication proteins Rep78, Rep68, Rep52, and Rep40. These Rep expression products have been shown to have many functions, including recognition, binding, and nicking of AAV DNA replication origins, DNA helicase activity, and transcriptional regulation from AAV (or other heterologous) promoters. The entire Rep expression product is required for AAV genome replication. A suitable homolog of the AAV rep coding region is the human herpesvirus 6 (HHV-6) rep gene, which is also known to mediate AAV2 DNA replication.

[0105]

[0106] AAV helper function can be introduced into host cells by transfecting the host cells with an AAV helper construct before or simultaneously with the transfection of an AAV expression vector. AAV helper constructs are therefore used to achieve at least transient expression of AAV rep and / or cap genes to compensate for the loss of AAV function necessary for productive AAV infection. AAV helper constructs lack AAV ITR and cannot replicate or package themselves. Such constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Several AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. Several other vectors encoding Rep and / or Cap expression products have also been described.

[0106]

[0107] The rAAV vectors, compositions, agents, drugs, and biological formulations (proteins) of the invention can be incorporated into pharmaceutical compositions, for example, pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions are particularly useful for in vivo administration and delivery to subjects.

[0107]

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. Methods and materials similar to or equivalent to those described herein may also be available in the practice or testing of the present invention, but preferred methods and materials are described herein.

[0108]

[0109] All applications, publications, patents, and other references cited herein, as well as citations from GenBank and ATCC, are incorporated herein by reference. In any conflict, this specification controls, including definitions.

[0109]

[0110] All properties disclosed herein can be combined in any combination. Each property disclosed herein may be replaced by an alternative property that serves the same, equivalent, or similar purpose. Accordingly, unless otherwise specified, the disclosed properties (e.g., modified nucleic acids, vectors, plasmids, recombinant AAV (rAAV) vectors, vector genomes, or rAAV virus particles) are examples of a genus of equivalent or similar properties.

[0110]

[0111] As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a bioacceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a substance that is not biologically or otherwise undesirable, for example, a substance that can be administered to a subject without causing a substantial degree of undesirable biological effect. Such a pharmaceutical composition may, for example, be used to administer rAAV vectors or rAAV particles to a subject.

[0111]

[0112] Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic and absorption enhancers or retarders, suitable for drug administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Auxiliary active compounds (e.g., antiseptics, antimicrobial, antiviral, and antifungal agents) can also be incorporated into the compositions.

[0112]

[0113] Pharmaceutical compositions include carriers, excipients, or additives suitable for administration by various routes. Compositions suitable for parenteral administration include aqueous and nonaqueous solutions, suspensions, or emulsions of the active compound, the preparations of which are generally sterile and may be isotonic with the blood of the intended recipient. Non-limiting examples include water, saline, dextrose, fructose, ethanol, animal oils, vegetable oils, or synthetic oils.

[0113]

[0114] The compositions, methods, and pharmaceutical compositions and delivery systems suitable for use of the present invention are known in the art (for example, Remington: The Science and Practice of Pharmacy (2003) 20th edition, Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th edition, Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th edition, Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th edition, Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al. Drug Delivery Systems(See 1980, RL Juliano (ed.), Oxford, NY, pp. 253–315).

[0114]

[0115] As used herein, “unit dose” or “unit dosage form” means a physically separated unit suitable for a unified administration to the subject being treated; each unit containing a predetermined amount is optionally associated with a pharmaceutical carrier (excipient, excipient, medium, or filler), but is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. A unit dosage form may be contained, for example, in ampoules and vials, and may be a liquid composition or a composition in a freeze-dried or lyophilized state; it may contain a sterile liquid carrier, which may be added, for example, before in vivo administration or delivery. Individual unit dosage forms may be contained in multi-dose kits or containers. rAAV vectors, rAAV particles, and their pharmaceutical compositions may be packaged in one or more unit dosage forms to facilitate administration and ensure dose uniformity.

[0115]

[0116] Various detection methods are available to confirm the state or improvement of a disease. Immunodetection methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradioactive assay, fluorescence immunoassay, chemiluminescence assay, bioluminescence assay, and Western blotting. Other methods are also known to those skilled in the art. A variety of useful immunodetection methods have been described in the scientific literature.

[0116]

[0117] Generally, immunoconjugation methods involve obtaining a sample suspected of containing Aβ protein and, optionally, contacting the sample with a primary antibody, i.e., a monoclonal or polyclonal specific to Aβ, under conditions effective in enabling the formation of an immune complex.

[0117]

[0118] Immunoconjugation methods include methods for detecting and / or quantifying the amount of Aβ protein in a sample, as well as detecting and / or quantifying any immune complexes formed during the binding process. Here, a sample suspected to contain Aβ protein can be obtained, the sample can be contacted with an antibody, and then the amount of immune complexes formed under specific conditions can be detected and quantified.

[0118]

[0119] The conditions necessary to enable the formation of immune complexes (primary immune complexes), and to contact a biological sample with antibodies for a sufficient period of time, generally involve simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the antibody to form an immune complex with any antigen it contains, i.e., to bind to it. After this, the sample-antibody composition, such as blood, plasma, or serum samples, or tissue sections, ELISA plates, dot blots, or Western blots, is typically processed (e.g., washed) to remove any nonspecifically bound antibody species, so that only the specifically bound molecules within the primary immune complex are detectable.

[0119]

[0120] In general, methods for detecting immune complex formation are well known in the art and can be achieved by applying a great many approaches. Such methods are commonly based on detecting any label or marker, such as radioactive, fluorescent, biological, and enzymatic tags. U.S. patents relating to the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Of course, as is well known in the art, the use of secondary binding ligands, such as a second antibody, and / or biotin / avidin ligand binding configurations can also be employed.

[0120]

[0121] As described above, the protein detection molecule (i.e., the binding ligand, e.g., antibody or antibody fragment) itself can be bound to a detectable label, in which case the label is simply detected, thereby enabling determination and / or quantification of the amount of primary immunocomplex in the composition. Alternatively, the first antibody bound within the primary immunocomplex can be detected by a second binding ligand having binding affinity to that antibody. In this case, the second binding ligand can be bound to a detectable label. The second binding ligand itself is often an antibody and is therefore sometimes called a “secondary” antibody. The primary immunocomplex is contacted with the labeled secondary binding ligand or antibody for a sufficient period of time under effective conditions that allow for the formation of a secondary immunocomplex. The secondary immunocomplex is then typically washed to remove all nonspecifically bound labeled secondary antibody or ligand, and the remaining label within the secondary immunocomplex is then detected.

[0121]

[0122] A further method involves the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody having binding affinity to the first binding ligand, is used to form a secondary immune complex as described above. After washing, the secondary immune complex is brought into contact again under conditions and for a period of time sufficient to allow the formation of an immune complex (tertiary immune complex) with a third binding ligand or antibody having binding affinity to the second antibody. The third ligand or antibody may be linked to a detectable label to enable the detection of the formed tertiary immune complex. This system may provide signal amplification if desired.

[0122]

[0123] As detailed above, immunoassays are binding assays. Specific immunoassays include various types of enzyme-linked immunosorbent assays (ELISAs) and / or radioimmunoassays (RIAs) known in the art. Immunohistochemical detection methods using tissue sections are also useful. Detection methods are not limited to such techniques, and / or Western blotting, dot blotting, FACS analysis, etc., are readily available.

[0123]

[0124] In a typical ELISA, an antibody is immobilized on a selected surface exhibiting protein affinity, such as a well in a microtiter plate. Next, a test composition suspected of containing AP protein, such as a clinical sample (e.g., a biological sample obtained from a subject), is added to the well. After washing to remove bound and / or nonspecifically bound immune complexes, the antibody-binding antigen can be detected. Detection is generally achieved by adding another (secondary) antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection may also be achieved by adding a second antibody, followed by a third antibody that has binding affinity to the second antibody and is linked to a detectable label.

[0124]

[0125] In another typical ELISA, a sample suspected of containing an antigen is immobilized on the well surface and / or subsequently comes into contact with a binder. After washing to remove bound and / or nonspecifically bound immune complexes, the bound anti-binding agent is detected. If the initial binder is linked to a detectable label, the immune complex may be detected directly. Alternatively, the immune complex may be detected using a second antibody that has binding affinity to the first binder and is linked to a detectable label.

[0125]

[0126] Another ELISA with immobilized antigens involves the use of antibody competition for detection. In this ELISA, labeled antibodies against the antigen are added to the wells, made binding-enabled, and / or detected by their labeling. The amount of antigen in an unknown sample is determined during the incubation period using the coated wells by then mixing the sample with the labeled antibody against the antigen. If the antigen is present in the sample, it acts to reduce the amount of antibody against the antigen available for binding to the wells, and therefore reduces the final signal. This is also suitable for detecting antibodies against an antigen in an unknown sample, where unlabeled antibodies also reduce the amount of antigen available for binding to the wells coated with the antigen and the labeled antibody.

[0126]

[0127] Regardless of the format used, ELISA has certain common characteristics, such as coating, incubation and binding, washing to remove nonspecifically bound species, and detection and / or quantification of bound immunocomplexes.

[0127]

[0128] In ELISA, it is perhaps more conventional to use secondary or tertiary detection methods rather than direct procedures. Therefore, after binding proteins or antibodies to the wells, coating with a non-reactive substance to reduce background noise, and washing to remove unbound material, the immobilized surface is brought into contact with the biological sample to be tested under conditions effective in enabling immune complex (antigen / antibody) formation. For the detection of immune complexes, then labeled secondary binding ligands or antibodies, and labeled tertiary antibodies or secondary binding ligands or antibodies associated with a third binding ligand are employed, and so on.

[0128]

[0129] "Conditions that enable immune complex (antigen / antibody) formation" means conditions that enable or promote binding. Such conditions may include diluting a sample, e.g., AP protein, τ oligomer, etc., and / or antibody composition with a solution such as BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween. Such added agents also tend to help reduce nonspecific background.

[0129]

[0130] "Suitable" conditions also mean that incubation takes place at a temperature or for a period of time sufficient to enable bonding. Typical, non-limiting incubation steps are generally about 1 to 2 to 4 hours, preferably at a temperature of around 25°C to 27°C, or overnight at around 4°C.

[0130]

[0131] After all incubation steps of the ELISA are completed, the contact surface is washed to remove non-complex-forming substances. Examples of washing procedures include washing with a solution such as PBS / Tween or borate buffer. Specific immune complexes are formed between the test sample and the first bound substance, and after subsequent washing, the amount of these immune complexes, even if trace amounts, can be determined.

[0131]

[0132] To provide a detection method, a second or third antibody may have a relevant label that enables detection. This may be an enzyme that causes color development when incubated with a suitable chromogenic substrate. Thus, for example, the first and second immunocomplexes can be contacted or incubated with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase-conjugated antibodies for a period and under conditions that favor further immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution, e.g., PBS-Tween).

[0132]

[0133] The amount of label is quantified by incubation with a labeled antibody, followed by washing to remove subsequent unbound material, and then incubation with a chromogenic substrate such as urea, bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or, in the case of peroxidase, H2O2, etc., as an enzyme label. Quantification is then achieved by measuring the degree of color produced, for example, using a visible spectrum spectrophotometer.

[0133]

[0134] As used herein, the singular forms "a," "and," and "the" include plural nouns unless otherwise specified in the context. Thus, for example, "a nucleic acid" includes multiple such nucleic acids, "a vector" includes multiple such vectors, and "a virus" or "particle" includes multiple such virions / particles.

[0134]

[0135] As used herein, all numbers or ranges of numbers include integers within that range, and fractions of numbers or integers within that range, unless otherwise explicitly stated in the context. Therefore, for illustrative purposes, "80% or more identity" includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so on.

[0135]

[0136] The terms "greater than" or "less than" integers include any number greater than or less than the number being referenced. For example, "less than 100" includes 99, 98, 97, etc., up to 1; and "less than 10" includes 9, 8, 7, etc., up to 1.

[0136]

[0137] As used herein, all numbers or ranges include fractions of numbers, integers within such ranges, and fractions of integers within such ranges, unless otherwise explicitly stated in the context. Therefore, for illustrative purposes, when we refer to a numerical range, for example, 1 to 10, it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and similarly 1.1, 1.2, 1.3, 1.4, 1.5, and so on. When we refer to a range, 1 to 50, it therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and the numbers up to 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, and similarly 2.1, 2.2, 2.3, 2.4, 2.5, and so on.

[0137]

[0138] When we refer to a series of ranges, it includes ranges that combine the numerical boundaries of different ranges within that series. Therefore, for explanatory purposes, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, 2,500 The ranges ~3,000, 3,000~3,500, 3,500~4,000, 4,000~4,500, 4,500~5,000, 5,500~6,000, 6,000~7,000, 7,000~8,000, or 8,000~9,000 include ranges such as 10~50, 50~100, 100~1,000, 1,000~3,000, 2,000~4,000, etc.

[0138]

[0139] The present invention is generally disclosed herein using affirmative language to describe a great many embodiments and aspects. The invention also includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, are excluded in whole or in part. For example, certain embodiments or aspects of the invention exclude materials and / or method steps. Therefore, while it is not generally stated herein what the invention does not include, aspects that are not explicitly excluded in the invention are nevertheless included in the disclosure herein.

[0139]

[0140] Several embodiments of the present invention have been described. Nevertheless, without departing from the spirit and scope of the invention, those skilled in the art can make various changes and modifications to the invention to make it suitable for various uses and conditions. Accordingly, the following embodiments are intended to illustrate the invention and do not limit the scope of the invention as claimed. [Examples]

[0140] Example 1 below is described in International Publication No. 2015 / 077473: Changes in the progression of amyloid deposition.

[0141] This example investigated changes in the progression of amyloid deposition in app / ps mice after overexpression of different ApoE isoforms via intracerebroventricular injection of adeno-associated virus serotype 4 (AAV4).

[0141]

[0142] The ε4 allele of ApoE (ApoEε4) is the primary genetic risk factor for Alzheimer's disease (AD), while the rarer ε2 allele of ApoE (ApoEε2) reduces this risk by about half. However, despite the discovery of such strong genetic clues nearly 17 years ago, the mechanism by which ApoE contributes to the risk remains unclear.

[0142]

[0143] To elucidate how different ApoE isoforms (ApoEε2, ε3, and ε4) affect the formation and stability of fibrous amyloid plaques, AAV4 vectors encoding each ApoE isoform were injected intravenously into 7-month-old APP / PS mice. In vivo multi-photon imaging was used to track populations of amyloid deposits at baseline and over two months after ApoE exposure, thus enabling a dynamic assessment of amyloidosis progression in living animals.

[0143]

[0144] The dynamics of amyloid plaque deposition were observed to differ depending on the isoform, with a 38% reduction in senile plaques in mice injected with ApoEε4 compared to those injected with ApoEε3 after 2 months. increase However, in mice treated with ApoEε2, the number of amyloid deposits was 15% lower. decrease This was observed. Postmortem analysis confirmed these results and revealed the presence of human ApoE protein decorating plaques in the cortex, reflecting both large-scale diffusion of the protein throughout the parenchyma and localized accumulation of the protein at sites where Aβ peptides were deposited. It is important to note that the increased content of this ApoEε4 protein was also associated with more severe synaptic loss around amyloid deposits.

[0144]

[0145] Overall, these data demonstrate that the overproduction of different ApoE isoforms can influence disease progression and that the range of synaptic loss, one of the parameters best correlated with cognitive impairment in AD patients, is moduloable.

[0145] 1. Intracerebroventricular injection of AAV4-ApoE induced stable expression of huApoE, enabling sustained detection of recombinant human ApoE (huApoE) protein in the brain.

[0146] In short, GFP and huApoE were immunodetected in APP / PS mice injected with the AAV4 vector. GFP signaling was observed in cells covering the entire ventricular area (upper panel) and the inner ventricular region, as well as in human APOE.

[0146]

[0147] To evaluate this approach, AAV4-Venus (control), -ApoE2, -ApoE3, and -ApoE4 were injected intravenously into wild-type mice. Two months after injection, human ApoE protein could be detected in the cortical parenchyma surrounding amyloid deposits (note the 3H1 antibody; only nonspecific background was observed in AAV4-GFP injected mice). Thus, significant levels of human ApoE were detected in the brain by ELISA, and immunohistochemical staining for Venus and ApoE confirmed the expression of different transgenes by cells lining the inner ventricular region.

[0147]

[0148] qRT-PCR experiments were performed to evaluate the mRNA levels of the transgene. From the standard curve, the concentration of huApoE mRNA could be determined based on the level of endogenous GAPDH. Samples were obtained from mice exposed for 2 or 5 months. An ELISA assay designed to specifically detect human APOE was performed on brain homogenates (Figure 1A, in International Publication No. 2015 / 077473). Low levels of recombinant protein could be detected in AAV4-APOE-injected mice compared to AAV4-GFP-treated animals, as quantified by a human APOE-specific ELISA (Figure 1B, in International Publication No. 2015 / 077473) and confirmed by Western blotting.

[0148] 2. Overexpression of each APOE isoform differentially affects the progression of amyloidosis.

[0149] In vivo two-photon imaging was used to track amyloid deposits over time in living animals. Specifically, APP / PS mice (7 months old) were stereotactically injected with ApoE2, ApoE3, ApoE4, and the AAV4 vector encoding Venus. One week later, a cranial fenestra was implanted, and after craniotomy, amyloid deposits were imaged over time. Two months later, the animals were killed, and postmortem analysis was performed.

[0149]

[0150] Two-photon images were prepared from APP / PS mice injected with AAV4-ApoE2, AAV4-ApoE3, or AAV4-ApoE4. Amyloid plaques could be detected after intraperitoneal injection of methoxy-X04 (5 mg / kg), and fluorescence angiography was obtained by injecting Texas Red Dextran (molecular weight 70,000 Da; 12.5 mg / ml sterile PBS) into the lateral tail vein. Images were acquired at 1 week (=T0), 1 month, and 2 months post-injection. The same field of view was acquired over time to track the progression of the lesions. Several new amyloid deposits appeared, but some were no longer detectable during the 2-month period.

[0150]

[0151] A complete analysis of in vivo imaging shows that the number of amyloid deposits increases significantly more rapidly in AAV4-ApoE4 injected APP / PS mice compared to both AAV4-ApoE3 and AAV4-Venus treated animals. In contrast, a slight but significant decrease in plaque density is measured when AAV4-ApoE2 is used (Figure 2, shown in International Publication 2015 / 077473). While plaques tend to become larger in AAV4-ApoE4 injected APP / PS mice (p<0.06), overall, plaque size remains constant. The summarized in vivo imaging data indicate that overexpression of each APOE isoform differentially affects the progression of amyloid deposition in vivo. Injection of AAV4-ApoE2 causes a slight decrease in amyloid density over time, while injection of AAV4-ApoE4 further exacerbates amyloidosis.

[0151] 3. The size of amyloid plaques varies depending on each ApoE isoform.

[0152] In vivo two-photon imaging allowed for tracking the size changes of each amyloid deposit over a two-month period. Plaque size remained stable or increased or decreased over time. The distribution of size ratios between T1 / T0 and T2 / T1 showed that mice injected with AAV4-ApoE4 tended to have larger amyloid plaques compared to other groups (Figure 3, shown in International Publication 2015 / 077473).

[0152] 4. Postmortem evaluation of amyloid load confirms the effects of ApoE2 and ApoE4 on amyloid deposition.

[0153] Two months after AAV4 injection, postmortem stereoanalytic evaluation revealed that animals injected with AAV4-ApoE4 had a higher density of amyloid plaques in the cortex, while no difference could be detected between the other groups (Figure 4A, shown in International Publication 2015 / 077473). This increase in the number of amyloid deposits was observed when the plaques were labeled with ThioS or Bam10. However, no change in the ratio between Bam10 and ThioS was detected. Five months after injection, the effect of each ApoE isoform was more pronounced compared to two months later (Figure 4B, as shown in International Publication 2015 / 077473). When mice were injected with AAV4-ApoE4, a significant increase in the density of deposits was observed, while the opposite effect was detected for ApoE2. Again, no change in the ratio between Bam10 and ThioS was detected.

[0153] 5. Each ApoE isoform differentially affects the synaptic density around amyloid deposits.

[0154] Array tomography was used to accurately determine the density of pre- and post-synaptic elements around amyloid deposits. This novel imaging method offers the ability to image tissue molecular structure at high resolution. Array tomography is based on ultrathin sectioning (70 nm), immunostaining, and 3D reconstruction of the sample. Representative images of array tomographic samples stained for amyloid plaques and the post-synaptic marker PSD95 are shown. Array tomographic images show a decrease in the number of post-synaptic markers PSD95 around amyloid deposits, but this effect disappears away from the plaque. Quantification of pre-synaptic markers (synapsin-1) and post-synaptic markers near and far from the plaque was determined in each group of mice injected with AAV4 (Figures 5A-D in International Publication 2015 / 077473). Extensive quantification of pre- and post-synaptic elements confirmed that decreased densities of synapsin 1 and PSD95 were associated with amyloid plaques, and that this effect was dramatically amplified when ApoE4 was overexpressed in the brains of APP / PS1 mice (Figures 5C and 5D, shown in International Publication No. 2015 / 077473). Overexpression of ApoE4 was associated with increased vertebral loss near amyloid deposits compared to other groups. Conversely, synapsin plaque density was higher around plaques in animals treated with ApoE2.

[0154] conclusion

[0155] Intravenous injection of AAV virus serotype 4 induced persistent and chronic overproduction of soluble recombinant protein throughout the cerebral parenchyma. Overexpression of ApoE2, ApoE3, and ApoE4 differentially affected the pathological course in APP / PS mice; therefore, injection of ApoE4 significantly increased the progression of amyloid loading compared to ApoE3. Conversely, ApoE2 was associated with a protective effect, with some amyloid deposits becoming undetectable two months after injection. Postmortem immunohistochemical analysis confirmed the adverse effects of ApoE4. Persistent overproduction of ApoE4 exacerbated synaptic loss around amyloid deposits compared to ApoE3, while the effect of ApoE2 was milder. This study demonstrated a direct in vivo correlation between ApoE isoforms, amyloidosis progression, and synaptic loss.

[0155] The following Example 2, described in International Publication No. 2015 / 077473, demonstrates therapeutic efficacy of CNS injury by CSF delivery: Treatment of CNS injury via cerebrospinal fluid (CSF) in large mammals.

[0156] To realize gene therapy for brain disorders, such as Alzheimer's disease, it was necessary to confirm whether therapeutic enzyme levels could be maintained at a steady state over the long term in mammals. It was discovered that target enzymes could be transduced into ependymal cells (cells lining the inner side of the ventricles in the brain) and secreted into cerebrospinal fluid (CSF). Adeno-associated virus (AAV4) was confirmed to be transduced into the ependymal cells of a mouse model with high efficiency (Davidson et al., PNAS, 28:3428-3432, 2000). In mice, substrate levels stored in the diseased brain were observed to normalize after AAV4 treatment.

[0156]

[0157] We investigated whether global delivery of the vector was feasible to achieve a steady state of enzyme levels within the CSF. First, it was necessary to find a vector that could transduce ependymal cells (cells lining the inner ventricles) in larger mammalian brains. Tests were conducted in canine and non-human primate models of LINCL. LINCL dogs are normal at birth but develop neurological signs around 7 months, testable cognitive impairment around 5-6 months, seizures around 10-11 months, and progressive vision loss.

[0157]

[0158] Adeno-associated virus (AAV) was selected as a vector because of its small size (20 nm) and the fact that most of its genetic material can be removed ("extracted") in a way that eliminates the viral gene and renders it non-replicable. The adeno-associated virus type 4 (AAV4) vector has been previously tested to determine if it can mediate global functional and pathological improvements in a mouse model of mucopolysaccharidosis type VII (MPS VII) caused by β-glucuronidase deficiency (Liu et al., J. Neuroscience, 25(41):9321-9327, 2005). Recombinant AAV4 vectors encoding β-glucuronidase were unilaterally injected into the lateral ventricle of MPS VII mice with established disease. The transduced ependyma expressed high levels of recombinant enzyme, and the secreted enzyme penetrated the cerebral and cerebellar structures, as well as the brainstem. Immunohistochemical studies revealed a close relationship between recombinant enzymes and the cerebral microvascular system, suggesting that β-glucuronidase reached the brain parenchyma via the perivascular space lining the inside of blood vessels. Aversive association learning was tested using contextual fear conditioning. Compared to age-matched heterozygous controls, affected mice exhibited impaired conditioned fear responses and contextual discrimination. This behavioral defect reversed 6 weeks after gene transfer in MPS VII mice treated with AAV4 β-glucuronidase. The data suggest that ependymal cells may play a role as a source of enzyme secretion to the perivascular and CSF regions of the brain parenchyma.

[0158]

[0159] Surprisingly, however, when this study was extended to larger mammals (i.e., dogs and non-human primates), the AAV4 vector was ineffective when targeting the ependymia of these animals. Rather, the AAV2 vector had to be used. In short, we created rAAV2 encoding TPP1 (AAV2-CLN2) and injected it intravenously to transduce it into the ependymia (Liu et al., J. Neuroscience, 25(41):9321-9327, 2005). TPP1 is enzyme-deficient in LINCL. The data suggested that ependymal transduction into the NHP brain resulted in a significant increase in the enzyme in the CSF. The results suggested elevated TPP1 activity levels in various brain regions (vertical axis shows the percentage of activity relative to the control) (Figure 7, shown in International Publication No. 2015 / 077473).

[0159]

[0160] In the first treated dog, vector delivery was not optimal, but CLN2 activity was still observed in the brain. The next dog received ICV delivery via stereotactic brain surgery. Cognitive abilities in the treated dogs were found to be significantly improved compared to the untreated dogs, as measured by T-maze performance (Figure 8, shown in International Publication 2015 / 077473). Furthermore, the effect of AAV2-CLN2 ICV delivery in the canine model of LINCL was very pronounced. Large ventricles were present in the untreated (- / -) animals, while the brains of the untreated control and treated animals did not show ventricles. After delivery of AAV.TPP1 to the ventricles of LINCL dogs, detectable enzyme activity was observed in various brain regions, including the cerebellum and upper spinal cord. In two additional surviving affected dogs, brain atrophy was significantly reduced, lifespan was extended, and cognitive function improved. Ultimately, this method demonstrates that in NHP, it is possible to achieve TPP1 activity levels 2 to 5 times higher than in the wild type.

[0160]

[0161] We created several AAV vectors and conducted tests to determine the optimal combination of ITR and capsid. We generated five different combinations and confirmed that AAV2 ITR was the most effective: AAV2 / 1 (i.e., AAV2 ITR and AAV1 capsid), AAV2 / 2, AAV2 / 4, AAV2 / 5, and AAV2 / 8. In large mammals (dogs and NHPs), we found that AAV2 / 2 functioned quite well, followed by AAV2 / 8, AAV2 / 5, AAV2 / 1, and AAV2 / 4. This was quite surprising, as the order of effectiveness of the viral vectors was the opposite of the order observed in mice.

[0161]

[0162] Therefore, this study demonstrated that cells lining the inner ventricular region of the brain can serve as a source of CSF-mediated recombinant enzymes for distribution throughout the brain, and that AAV2 / 2 is an effective medium for administering therapeutic agents, such as the gene encoding CLN2(TPP1), in dogs and non-human primates.

[0162] Example 3 below, described in International Publication No. 2015 / 077473, demonstrates the effects of different ApoE isoforms delivered to the CNS by an AAV vector: Human APOE isoforms delivered by gene transfer differentially modulate Alzheimer's disease by affecting amyloid deposition, efflux, and neurotoxicity.

[0163] Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder and a major public health concern. Among the susceptibility genes associated with late-onset and sporadic forms of AD, the apolipoprotein Eε4 (APOE gene; ApoE protein) allele is exceptionally important as a genetic risk factor. Even one copy of APOEε4 substantially triples the risk of developing the disease compared to the most common APOEε3 allele, while two copies increase it twelvefold. Interestingly, APOEε2 has the opposite effect and is also a protective factor; the genetic makeup of this particular allele reduces the age-adjusted risk of AD by about half compared to APOE3 / 3. The mean age of onset of dementia also corresponds to these risk profiles: APOE4 / 4 carriers develop the disease in their mid-60s, APOE2 / 3 carriers in their early 90s (almost a 30-year difference), while APOE3 / 3 individuals develop it somewhere in between, in their mid-1970s.

[0163]

[0164] The mechanisms by which ApoE influences AD are debatable. Since all known genes involved in the rare autosomal dominant form of the disease are involved in Aβ peptide production, Aβ accumulation containing senile plaques in the hippocampus and cortex of patients is thought to play a central role in AD. Interestingly, APOE genotype has been shown to strongly influence the degree of amyloid deposition in AD patients, as well as the amount of neurotoxic soluble oligomeric Aβ detected in autopsy samples. ApoE isoforms are suggested to differentially affect cerebral vascular integrity, influence the efflux of Aβ peptides across the blood-brain barrier, and thus regulate the accumulation of perivascular amyloid aggregates (cerebral amyloid angiopathy or CAA). Furthermore, ApoE is also directly involved in neurodegeneration and neuroplasticity. In these contexts, the effects of ApoE2 have not been extensively investigated.

[0164]

[0165] Genetically engineered animals expressing human APOE2, -E3, and -E4 exhibited a similar ranking of amyloid load to humans, which is consistent with the hypothesis that different ApoE isoforms influence plaque initiation and / or growth. However, further testing is needed to investigate the mechanisms of ApoE-mediated effects on existing amyloid deposits and pre-existing neurodegeneration. To overcome this knowledge gap, we used a gene transfer approach in which adeno-associated virus vectors expressing various APOE alleles (or GFP controls) were injected into the lateral ventricles, primarily transducing them to the ependyma, which then acts as a biofactory delivering ApoE into the cerebrospinal fluid and interstitial fluid. Next, in vivo multiphoton microscopy was used to track the effects of various ApoE isoforms on plaque formation, growth, and, in the case of ApoE2, lysis; an in vivo microdialysis approach was used to monitor the biochemical changes of ApoE and Aβ in ISF; and array tomography was used to evaluate changes in Aβ-related neurotoxicity.

[0165]

[0166] ApoE isoforms influence the level of soluble oligomeric Aβ in ISF, the rate of Aβ fibrillation and deposition, the stability of amyloid deposits once formed, their elimination, and the degree of neurotoxic effects around plaques. In fact, AD mice treated with ApoE4 showed increased amounts of soluble Aβ, denser fibrous plaques, exacerbated synaptic element loss, and an increased number of neurite dystrophy around each deposit, while ApoE2 showed a relative protective effect. These data support the hypothesis that the APOE allele mediates its effects on AD primarily through Aβ, highlighting ApoE as a potential therapeutic target.

[0166] result Intraventricular injection of AAV4-APOE stabilizes APOE expression in the brain, leading to sustained production of human ApoE.

[0167] Apolipoprotein E is a naturally secreted protein, primarily produced by astrocytes and microglia cells, and can diffuse throughout the brain parenchyma. The inventors utilized the advantages of this property by injecting GFP (control) or AAV serum type 4 encoding each APOE allele into the lateral ventricles of 7-month-old APP / PS1 mice. Given that the cerebral region is affected by characteristic lesions of AD, this strategy offered significant advantages compared to multiple intraparenchymal injections.

[0167]

[0168] Two months after injection, transduced cells were detected in the choroid plexus and the ependyma lining the ventricles, thus confirming the functionality of the AAV4 vector. Both human and mouse ApoE proteins were detected using species-specific antibodies by ELISA (Figures 9A, 9B, and 15A in International Publication No. 2015 / 077473) and Western blotting. We observed an average human apolipoprotein E concentration of 20 μg per 1 mg of total protein (Figure 9A in International Publication No. 2015 / 077473), which accounts for approximately 10% of endogenous mouse apoE (Figure 9B in International Publication No. 2015 / 077473). This small additional amount of human ApoE did not detectably alter the level of endogenous mouse apoE protein (Figure 15A in International Publication No. 2015 / 077473). A small but statistically significant decrease was observed 2–5 months after AAV4 injection (Figure 15B, shown in International Publication No. 2015 / 077473). Nevertheless, human protein levels remained detectable compared to the control group, suggesting that AAV4-mediated transduction provided a sustained production platform for secreted recombinant protein throughout the entire substance. Indeed, human ApoE protein was detectable around amyloid deposits in APP / PS1 mice, throughout the cortical mantle where endogenous mouse ApoE protein is known to accumulate.

[0168]

[0169] Next, we evaluated the presence of human ApoE in the interstitial fluid (ISF), an extracellular compartment that also contains highly biologically active Aβ-soluble species. Since the amount of ApoE detected in whole brain lysates is relatively small, each AAV4-APOE vector was injected into several apoE KO mice, and the presence of the human protein was tracked using highly sensitive but non-species-specific antibodies. Microdialysis techniques were used to confirm the presence of ApoE in the ISF of the apoE KO injected animals.

[0169]

[0170] Overall, these data confirm that a single intracerebroventricular injection of AAV4 was sufficient to cause sustained production of the protein of interest throughout the brain parenchyma and in the ISF, and that the ependyma / chroid plexus can be used as a "biological pump" to deliver therapeutically useful proteins to the brain.

[0170] Infusion of ApoE isoforms differentially affects amyloid peptide and plaque deposition

[0171] Vectors expressing GFP or various ApoE isoforms were transduced into APP / PS1 mice, and the mice were euthanized five months later. Analysis of amyloid plaque load revealed that after five months, a significant increase in amyloid deposition density was observed in the cortex of animals injected with AAV4-APOE4 compared with animals expressing APOE2. Plaque densities in AAV4-GFP and AAV4-APOE3 treated mice were at intermediate levels and did not differ from each other (Figure 16A shown in International Publication No. WO 2015 / 077473).

[0171]

[0172] Aβ measured from formic acid extracts 40 and Aβ 42 peptide concentrations were similar to the changes observed for amyloid plaque content, such that after five months, an increase in amyloid peptide concentration was found in mice expressing the APOE4 allele (Figure 16B shown in International Publication No. WO 2015 / 077473), whereas the opposite effect was detected with APOE2. Aβ in the TBS soluble fraction 40 and Aβ 42 peptide content was similarly affected by injection of each AAV-APOE (Figure 16C shown in International Publication No. WO 2015 / 077473). Furthermore, the ratio between aggregated and soluble Aβ peptides remained unchanged upon exposure to ApoE, thus suggesting that overexpression of different human ApoE isoforms simultaneously regulates both fibrillar and soluble amyloid species.

[0172]

[0173] Overexpression of each ApoE isoform for only two months produced a smaller effect than that observed in the five-month study. Nevertheless, a significant increase in amyloid plaque density was observed in the cortical regions of AAV4-APOE4 injected mice compared to other experimental groups (Figure 16A, International Publication 2015 / 077473). This was comparable to the amount of AP contained in the formic acid fraction (Figure 16C, International Publication 2015 / 077473), demonstrating the superior effect of this particular variant. When AAV4-APOE2 or AAV4-APOE4 was expressed for two months, TBS-soluble Aβ 40 / 42 Only the species showed a tendency to be either lower or higher (data not shown).

[0173]

[0174] To confirm whether the presence of human ApoE isoforms could reflect their initial changes in the degree of Aβ fibrosis, the ratios between strong immunostaining of AP were also measured two months after injection using Bam10 (labeling all amyloid deposits) and Thio-S (staining only high-density cores). No changes were detected among the three isoforms, suggesting that there was no differential effect on the distribution of high-density, diffusible amyloid deposit populations across the experimental group at this timeframe (Figure 16B, shown in International Publication 2015 / 077473). These data indicate that longer-term exposure to ApoE variants has a stronger effect on amyloid deposition than shorter-term exposure.

[0174]

[0175] ApoE has been suggested to play a role in Aβ transport across the blood-brain barrier. To test whether exposure to ApoE isoforms can regulate the efflux of AP peptides across the blood-brain barrier, we investigated the plasma of each injected animal containing Aβ 40The concentrations were measured. It was observed that the plasma content of human Aβ in mice injected intracerebroventricularly with AAV4-APOE3 and AAV4-APOE4 was lower than that in mice injected with AAV4-APOE2 and AAV4-GFP (Figure 10D, shown in International Publication No. 2015 / 077473). This suggests that both E3 and E4 variants help retain AP within the central nervous system compartment, and is consistent with previous data suggesting a relative increase in Ap concentration in the brain parenchyma and that the half-life of AP is enhanced due to ApoE.

[0175]

[0176] Recent studies have shown that APOE4 carriers are more susceptible to neurovascular dysfunction, and that blood-brain barrier disruption is frequently observed in APOE4 transgenic mice, even in the absence of amyloid deposition. To evaluate whether intracerebroventricular injection of AAV4-APOE in APP / PS mice could impair BBB integrity, postmortem staining with Prussian blue was performed. Although several hemosiderin-positive local areas were sparsely distributed throughout the brain in all groups, no significant differences were observed between the animal experimental groups.

[0176] Expression of ApoE isoforms regulates the dynamics of amyloidosis progression.

[0177] After 5 months, ApoE4 was associated with an increase in amyloid deposit density, while the opposite effect was observed with ApoE2. This may reflect changes in the rate of amyloid-beta deposition, efflux, or both. To assess how ApoE variants affect the dynamic progression of amyloidosis, we used in vivo two-photon imaging to track the dynamics of amyloid plaque formation and efflux. Mice were injected intraventricularly with the AAV4 vector at 7 months of age, and a cranial window was implanted one week after injection for the first imaging session (T0). Amyloid deposits were imaged within the same field of view at 1 month (T1) and 2 months (T2). After the second imaging session, the mice were euthanized for postmortem analysis.

[0177]

[0178] While the majority of amyloid deposits remained stable, incidental new plaques were detected within small visual volumes over a two-month period. Furthermore, although very rare, methoxy-positive plaques imaged at the start of the experiment were not detected after one or two months, suggesting that some plaques may have been shed. Over time, the overall volume density of amyloid deposits increased, with the density at T2 being an average of 23% higher than that at T1. After two months, APP / PS1 mice treated with ApoE4 showed a faster rate of amyloid progression, while animals exposed to ApoE2 showed a significantly reduced amyloid deposit density compared to GFP (0.66), ApoE3 (0.67), and ApoE4 (0.74) (Figures 11A, 11B in International Publication 2015 / 077473). Importantly, the changes in ApoE2 reflect a decrease from baseline, directly and for the first time, demonstrating active plaque efflux without immune intervention. In contrast to data obtained from APOE transgenic animals, these results demonstrate that even after amyloid deposition has already begun, inducing a slight increase in ApoE levels can influence the ongoing amyloidogenesis process.

[0178]

[0179] Next, the growth of individual amyloid plaques was evaluated by measuring the T1 / T0 and T2 / T1 ratios for the cross-sectional area of ​​each deposit. While differences between groups were detected in T1 (T1 / T0 ratio), none were detected in T2 (T2 / T1 ratio, Figure 12 in International Publication No. 2015 / 077473), suggesting that the presence of human ApoE variants primarily affects plaque growth during the first month after exposure, but there is no difference in this parameter thereafter. In particular, in ApoE4-treated mice, the size of amyloid deposits grew significantly larger compared to both ApoE2 and ApoE3, suggesting that not only the number of plaques but also their size was exacerbated by this allele. Therefore, ApoE4 affects both AP peptide seeding and the size of existing plaques.

[0179] Synaptic density around amyloid deposits is exacerbated by ApoE3 and ApoE4 isoforms compared to ApoE2.

[0180] Synaptic loss is the parameter that best correlates with cognitive impairment. We recently revealed that the presence of ApoE4 is associated with higher levels of synaptic oligomer Aβ in the brains of human AD patients, and that it causes a significant decrease in synaptic density around amyloid plaques compared to ApoE3 (RM Koffie et al., Apolipoprotein E4 effects in Alzheimer's disease are mediated by synaptotoxic oligomeric amyloid-beta. Brain 135, 2155 (Jul, 2012); T. Hashimoto et al., Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid beta peptides. J Neurosci 32, 15181 (Oct 24, 2012)). Furthermore, recent in vitro evidence has demonstrated that ApoE4 is unable to protect against Aβ-induced synaptic loss (M. Buttini et al., Modulation of Alzheimer-like synaptic and cholinergic deficits in transgenic mice by humanapolipoprotein Edepends on isoform, aging, and overexpression of amyloid betapeptides but not plaque formation. J Neurosci 22, 10539 (Dec 15, 2002); A. Sen, DL Alkon, TJ Nelson, Apolipoprotein E3 (ApoE3) but not ApoE4 protects against synaptic loss through increased expression of protein kinase C epsilon. J Biol Chem 287, 15947 (May 4, 2012)).Therefore, we hypothesized that the continuous and widespread distribution of each ApoE isoform can differentially influence not only the dynamics of Aβ deposition and efflux in the brains of APP / PS mice, but also the integrity of synapses around amyloid deposits.

[0180]

[0181] Using array tomography, a high-resolution technique based on immunofluorescence staining of ultrathin tissue sections, the densities of pre- and postsynaptic elements (synapsin-1 and PSD95, respectively) were determined (KD Micheva, SJ Smith, Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron55, 25 (Jul 5, 2007); RM Koffie et al., Oligomeric amyloidbeta associates with postsynaptic densities and correlates with excitatorysynapseloss near senile plaques. Proc Natl Acad Sci USA, 106, 4012 (Mar 10, 2009)). Since amyloid oligomer species have been shown to be highly concentrated near amyloid deposits, we used established protocols to quantify synapsin-1 and PSD95 spots far (more than 50 μm) or near (less than 50 μm) plaques (RM Koffie et al., Oligomeric amyloidbetaassociates with postsynaptic densities and correlates with excitatory synapseloss near senile plaques. Proc Nall Acad Sci USA 106, 4012 (Mar 10, 2009)). When APOE3 or APOE4 was expressed, we observed an exacerbation of presynaptic element loss near plaques, but this was not the case after injection of AAV4-APOE2 or AAV4-GFP (Figure 13A in International Publication No. 2015 / 077473).In contrast, while the density of postsynaptic plaques remained unchanged among mice injected with GFP, ApoE2, and ApoE3, ApoE4-treated animals showed significant loss of PSD95 around amyloid deposits, thus enhancing the adverse effect of ApoE4 on the neurotoxic effects of Aβ (Figure 13C, International Publication 2015 / 077473). When the density of synaptic elements was evaluated in areas far from amyloid deposits (more than 50 μm), no difference was detected between the groups, suggesting that while human ApoE variants themselves do not have an effect on synaptic density, ApoE isoforms have a significant effect on Aβ-induced neurotoxicity. Therefore, the relative synaptic loss observed in ApoE3 and ApoE4 is directly related to the presence of Aβ peptides around each plaque (at a distance of less than 50 μm from its edge).

[0181]

[0182] As an additional neuropathological parameter, the number of amyloid deposit-associated neuritis dystrophy in APP / PS1 mice injected with AAV4 was also evaluated. In addition to a decrease in spine density around the deposits, senile plaques evoke more common changes such as neuropils with increased neurite curvature and the appearance of expansive dystrophy. These pathological changes are likely due to soluble oligomeric Aβ species enriched in the region within 50 μm of the plaque surface. Overexpression of ApoE4 was observed to exacerbate the formation of amyloid deposit-associated SMI312-positive neuritis dystrophy compared to GFP, ApoE2, and ApoE3 (Figure 13C, shown in International Publication 2015 / 077473). These results confirm the observation that the ApoE4 isoform has the most potent effect, not only regulating plaque formation but also affecting amyloid-related neurotoxicity.

[0182] Human ApoE protein alters the amount of oligomeric Aβ species in the interstitial fluid of another mouse model of AD.

[0183] Next, we addressed the question of whether the presence of different ApoE isoforms within the ISF could alter the amount of soluble amyloid species contained in the same extracellular compartment. To validate our previous findings in different transgenic mouse lines, we chose to inject Tg2576 mice, another AD model. Tg2576 mice overexpress a mutant form of APP containing the Swedish mutation and exhibit a much milder phenotype than APP / PS1 mice at a given age. When injected into a cohort of 16-18 month-old animals, amyloid deposits were already present at the time of AAV4-APOE transduction. Three months after gene transfer, microdialysis probes were inserted into the hippocampus, samples were collected, and initial changes associated with each APOE variant within the ISF were characterized.

[0183]

[0184] After injection of AAV4-APOE4, the concentration of Aβ oligomer species, as measured using a specific 82E1 / 82E1 ELISA assay, was observed to be significantly higher (42±7%) compared to the case of AAV4-APOE2 (Figure 14, shown in International Publication No. 2015 / 077473). This suggests that the presence of ApoE can modulate the properties of amyloid aggregates in this extracellular compartment. Furthermore, in ISF, Aβ 40 and Aβ 42 An overall evaluation revealed the same trend, but it was not statistically significant (Figure 17A in International Publication No. 2015 / 077473), suggesting that the presence of different ApoE isoforms within the ISF has a slightly greater effect on the aggregation state of amyloid peptides than the overall amount.

[0184]

[0185] As expected, postmortem biochemical analysis of brains obtained from Tg2576 mice exposed to various ApoE isoforms showed that in ApoE4-treated animals, Aβ in the formic acid fraction was higher. 42A significant increase in concentration was observed (Figure 17B in International Publication No. 2015 / 077473), confirming the inventors' observations in APP / PS1 mice in a second transgenic model.

[0185]

[0186] In summary, these biochemical indicators suggest that ApoE expression in Tg2576 mice induces changes in amyloid biology similar to those observed in APP / PS1 mice. Importantly, when such neurotoxic species can directly interact with synaptic terminals, initial changes have been observed in oAβ content within ISF.

[0186] Example 4: Conclusions derived from Examples 1-3

[0187] The above studies in Examples 1-3 are described in International Publication No. 2015 / 077473 and demonstrate the therapeutic efficacy of administering an AAV vector containing a transgene encoding a protective ApoE isoform into the CNS of an animal model of Alzheimer's disease. Therefore, these studies support the suggestion that protective ApoE isoforms delivered to the CNS by an AAV vector could be a therapeutic agent for Alzheimer's disease.

[0187] Example 5: Representative assay method for detecting ApoE APOE ELISA

[0188] A specific ELISA assay was used to detect both human APOE protein and endogenous mouse APOE protein. In short, ELISA plates were coated overnight with either 1.5 μg / ml goat anti-APOE antibody (to detect mouse APOE) or 1.5 μg / ml WUE4 antibody (to detect human APOE), and blocked at 37°C for 1.5 hours with 1% skim milk diluted in PBS. Human recombinant apoE protein (for human-specific assays, Biovision) or a mouse standard prepared in-house from brain extract (for mouse-specific assays) was used as the standard, and samples were diluted in ELISA buffer (0.5% BSA and 0.025% Tween 20 dissolved in PBS) and incubated overnight. After washing, human-specific (goat-apoe Millipore; 1:10,000) or mouse-specific (Abcam ab20874; 1:2,000) detection antibodies were used, and the samples were incubated for 1.5 hours with the corresponding HRP-conjugated secondary antibody. After developing the signal using a TMB substrate, the solution was stopped using H3PO4. The colorimetric analysis results were measured at 450 nm.

[0188] Quantification of Aβ

[0189] Aβ 40 and Aβ 42 The concentration of BNT-77 / BA-27(Aβ) should be adjusted according to the manufacturer's instructions. 40 (For use) and BNT-77 / BC-05(Aβ 42The measurements were performed using a sandwich ELISA (Wako). Aβ oligomers were quantified using an 82E1 / 82E1 sandwich ELISA (Immuno-Biological Laboratories) with the same N-terminal (residues 1-16) antibody for both capture and detection (W. Xia et al., A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease. Arch Neurol 66, 190 (Feb, 2009)).

[0189] Example 6: Representative AAV capsid sequences AAV-LK03 VP1 capsid (SEQ ID NO: 1): [ka]

[0190] Amino acid sequence of AAV4-1 VP1 capsid (SEQ ID NO: 2): [ka]

[0191] Amino acid sequence of AAV4-1 VP2 capsid (SEQ ID NO: 3): [ka]

[0192] Amino acid sequence of AAV4-1 VP3 capsid (SEQ ID NO: 4): [ka]

[0193] [Related applications]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 383,274, filed 2 September 2016. The entire contents of the above application, including all text, tables, and arrangements, are incorporated herein by reference.

Claims

1. The use of rAAV particles and vectors for the manufacture of pharmaceuticals for the treatment of Alzheimer's disease in mammals, wherein the mammals are at risk of having Alzheimer's disease, and the mammals at risk of having Alzheimer's disease have the ApoEε4 allele and have a disrupted blood-brain barrier. The rAAV particles contain the AAV capsid protein, and the vector contains nucleic acids encoding a protective human ApoE isoform, inserted between a pair of AAV terminal inverted repeat sequences. The pharmaceutical product is intended for administration to non-central nervous system (CNS) cells, organs, or tissues of a mammal in a manner effective in infecting the non-CNS cells, organs, or tissues within the mammal so that the CNS cells, organs, or tissues express and secrete the protective human ApoE isoform during the mammalian circulation, which is used to deliver the protective human ApoE isoform to the central nervous system of the mammal, wherein the protective human ApoE isoform is at least 90% identical to ApoEε2.

2. The use of rAAV particles and vectors for the manufacture of pharmaceuticals for the treatment of Alzheimer's disease in mammals, wherein the mammals are at risk of having Alzheimer's disease, and the mammals at risk of having Alzheimer's disease possess the ApoEε4 allele and have a disrupted blood-brain barrier. The rAAV particles contain the AAV capsid protein, and the vector contains nucleic acids encoding a protective human ApoE isoform between a pair of AAV terminal inverted repeat sequences. The pharmaceutical agent is intended for administration to non-central nervous system (CNS) cells, organs, or tissues of a mammal in a manner effective for infecting liver cells within the mammal so that the liver cells express and secrete the protective human ApoE isoform during the mammalian circulation, which is then delivered to the mammalian central nervous system, wherein the protective human ApoE isoform is at least 90% identical to ApoEε2.

3. The use according to claim 1 or 2, wherein the protective human ApoE isoform is expressed in an amount that produces a beneficial or therapeutic effect on the mammal.

4. The use according to claim 1 or 2, wherein the protective human ApoE isoform is expressed in an amount that produces a beneficial or therapeutic effect on the mammal with respect to the physical, physiological, CNS / brain pathophysiological, biochemical, histological, or behavioral characteristics of CNS disease.

5. The use according to any one of claims 1 to 4, wherein the protective human ApoE isoform is human ApoEε2.

6. The use according to any one of claims 1 to 5, wherein the nucleic acid encoding the protective human ApoE isoform has reduced CpG compared to a nucleic acid in which the cytosine-guanine dinucleotide (CpG) encoding the protective human ApoE isoform is not reduced.

7. The use according to any one of claims 1 to 6, wherein the vector further comprises one or more of the introns, expression regulatory elements, filler polynucleotide sequences, and / or polyA signals, or a combination thereof.

8. The use according to any one of claims 1 to 7, wherein the pair of ITRs are derived from or include any sequence or mixture thereof of ITRs AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, and LK03.

9. The use according to any one of claims 1 to 8, wherein the AAV capsid protein is derived from or comprises any sequence or mixture thereof from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV4-1, AAV-2i8.

10. The use according to claim 1, wherein the non-CNS cells, organs, or tissues include mammalian endocrine cells, organs, or tissues.

11. The use according to claim 1, wherein the non-CNS cells, organs, or tissues include liver cells, liver or liver tissue, and pancreatic cells, pancreas or pancreatic tissue.

12. The use according to claim 1, wherein the non-CNS cells, organ, or tissue includes hepatocytes of the liver or islets of the pancreas.

13. The use according to claim 2, wherein the liver cells include hepatocytes.

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