Route of administration

Abstract

The present invention relates to the treatment of neurodegenerative diseases via an intramucosal administration route using recombinant adeno-associated virus (rAAV) particles containing an AAV true type (AAVTT) capsid and heterologous nucleic acids packaged therein.

Description

[Technical Field]

[0001] The present invention relates to the field of administration routes for recombinant adeno-associated virus (rAAV) particles, namely AAV true type (AAVTT) capsids and heterogeneous nucleic acids packaged therein, for use in the treatment of neurodegenerative diseases, and to pharmaceutical compositions comprising such particles. [Background technology]

[0002] Neurodegenerative diseases affect both adult and pediatric patients. They are often progressive and follow a relentless course, leading to structural and functional degeneration of the central and peripheral nervous systems. A common feature of many of these diseases is a complex and severe impairment of cognitive, motor, and sensory functions, resulting in a loss of quality of life and ultimately death. Because neurodegenerative diseases vary in etiology, prevalence, diagnosis, and management, the patient population is heterogeneous.

[0003] Treatment for neurodegenerative diseases is limited to symptomatic treatment, and there are still no disease-modifying therapies available that can treat the disease by reversing, stopping, or delaying disease progression, eliminating symptoms, or even curing the disease.

[0004] Gene therapy holds great potential for treating neurodegenerative diseases, and numerous preclinical and clinical studies are currently underway to address this need.

[0005] Adeno-associated viruses (AAVs) are a primary option in clinical trials focused on the central or peripheral nervous system. Numerous factors make AAVs ideal as gene delivery vehicles for neurodegenerative diseases. AAV-based vectors are particularly promising in most cases because they exhibit low immunogenicity, a low risk of insertional mutagenicity, and can mediate long-term gene expression in both dividing and non-dividing cells, which is necessary for neurodegenerative diseases (Ojala DS et al., 2015).

[0006] Systemic administration routes are particularly unsuitable for AAV-based gene therapies for the treatment of neurodegenerative diseases. To date, intracranial administration has been the common route for delivering AAV-based gene therapies to the brain (Wood et al., 2022). This approach bypasses biological transport barriers (the so-called "blood-brain barrier") and further reduces the risk of vector neutralization by circulating antibodies. Intracranial administration has significant drawbacks. Due to poor vector diffusion, the expression of the transgene is limited to the vicinity of the administration site, which is a major disadvantage for diseases affecting multiple areas of the central nervous system. For example, AAV2 has particularly strong neuronal tropism (Bartlett JS et al., 1998) and has been supported in clinical trials due to its established safety record. However, other serotypes such as AAV1, AAV5, or AAV9 have been shown to be highly efficient in transducing neurons (Mandel RJ et al., 2004; Sawamoto K. et al., 2018; Wang D. et al., 2019).

[0007] Frontotemporal dementia (FTD) is a fatal neurodegenerative disease that typically presents with impairments in executive function, behavior, language, or comprehension. These symptoms are associated with a characteristic pattern of brain atrophy affecting subcortical regions such as the prefrontal and temporal cortex, thalamus, and hippocampus (Whitwell JL et al., 2011; Liscic RM, 2017). Patients universally exhibit a progressive course, with an average survival time of 8 years from the onset of symptoms (Coyle-Gilchrist IT et al., 2016). In 5–10% of FTD patients, pathogenic loss-of-function mutations can be identified in the granulin (GRN) gene, which encodes progranulin (PGRN), a common lysosomal protein (Rohrer JD et al., 2009). GRN mutations can present with the clinical features of other neurodegenerative diseases, such as progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease, Lewy body dementia, Alzheimer's disease (Le Ber I. et al., 2008), mucopolysaccharidosis (MPS), neuronal ceroid lipofuscinosis (NCL), progressive neurodegenerative disorder characterized by intracellular accumulation of ceroid lipo dyes (Kamate M. et al., 2019), and amyotrophic lateral sclerosis (ALS), sometimes with dementia (ALS-D) (Irwin D. et al., 2009).

[0008] AAV-based gene therapies to correct GRN mutations and thus provide patients with sufficient levels of PGRN have been recently reported (International Publication No. 22034130; International Publication No. 2019070894; International Publication No. 2019070894). In particular, intracardiac delivery has been reported as a preferred route of administration for GRN AAV-based gene therapy (International Publication No. 2017151884). More recently, intracisional cisternostomy (ICM) AAV1- or AAV9-based gene therapies have also been reported (International Publication No. 22046988; ClinicalTrial.gov NCT04747431 and NCT04408625) (International Publication No. 22046988).

[0009] Rodent and non-human primate surrogates remain invaluable models for developing gene therapies for human use. Rodent models are still crucial not only for identifying potential treatment targets but also for exploring pathways and disease mechanisms. However, because primate neural structures are similar to (and in some cases nearly identical to) human neural structures, the use of primate models remains the only meaningful approach to investigating which brain regions should be successfully targeted by specific viral vectors in order to establish the most effective administration routes for neurodegenerative diseases in human use (Wozar F. et al., 2022; Pignataro D. et al., 2018; FDA draft guidance 2022).

[0010] Therefore, there remains a need to identify the most effective routes of administration for disease-modifying therapies (such as AAV-based gene therapy) for neurodegenerative diseases affecting large central nervous system regions, such as FTD (including adult-onset neurodegenerative diseases). In particular, there remains a need to identify the most effective routes of administration for treating GRN mutations with AAVtt-based gene therapy. [Overview of the project]

[0011] In a first aspect, the present invention provides a method for treating neurodegenerative diseases in primates by administering a therapeutic dose of recombinant adeno-associated virus (rAAV) particles to primates, wherein the rAAV particles comprise a) an AAV true type (AAVTT) capsid comprising or comprising SEQ ID NO: 1, and b) heterogeneous nucleic acids packaged therein, the heterogeneous nucleic acids comprising i) a 5' reverse terminal repeat (ITR) or fragment thereof and a 3' ITR or fragment thereof; ii) a transgene; and iii) one or more regulatory sequences that direct the expression of the transgene, and the treatment comprises administering the rAAV particles to the putamen of the primate.

[0012] In a second aspect, the present invention relates to a method for delivering a therapeutic dose of recombinant adeno-associated virus (rAAV) particles to the central nervous system of a primate, wherein the rAAV particles comprise a) an AAV true-type capsid comprising or consisting of SEQ ID NO: 1, and b) heterogeneous nucleic acids packaged therein, the heterogeneous nucleic acids comprising i) a 5' reverse-terminal repeat (ITR) or a fragment thereof and a 3' ITR or a fragment thereof; ii) a transgene; and iii) one or more regulatory sequences that direct the expression of the transgene, and the method comprises the step of administering the rAAV particles to the putamen of the primate.

[0013] In a third aspect, the present invention provides recombinant adeno-associated virus (rAAV) particles for use in the treatment of neurodegenerative diseases in primates, wherein the rAAV particles comprise a) an AAV true type (AAVTT) capsid comprising or comprising SEQ ID NO: 1, and b) heterogeneous nucleic acids packaged therein, the heterogeneous nucleic acids comprising i) a 5' reverse terminal repeat (ITR) or a fragment thereof and a 3' ITR or a fragment thereof; ii) a transgene; and iii) one or more regulatory sequences that direct the expression of the transgene, and the rAAV particles are administered to the putamen of the primate.

[0014] In a fourth aspect, the present invention relates to recombinant adeno-associated virus (rAAV) particles for use in the delivery of heterologous nucleic acids to the central nervous system of a primate, wherein the rAAV particles comprise a) an AAV true type (AAVTT) capsid comprising or comprising SEQ ID NO: 1, and b) a heterologous nucleic acid to be delivered, packaged therein, the heterologous nucleic acid comprising i) a 5' reverse terminal repeat (ITR) or a fragment thereof and a 3' ITR or a fragment thereof; ii) a transgene; and iii) one or more regulatory sequences that direct the expression of the transgene, and the rAAV particles are administered to the putamen of the primate.

[0015] In a fifth aspect, the present invention describes a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles and one or more carriers and / or excipients, wherein the rAAV particles comprise a) an AAV true-type capsid comprising or comprising SEQ ID NO: 1, and b) heterogeneous nucleic acids packaged therein, the heterogeneous nucleic acids comprising i. 5' inverted end repeats (ITRs) or fragments thereof and 3' ITRs or fragments thereof; ii. a transgene; and iii. one or more regulatory sequences that direct the expression of the transgene, and the pharmaceutical composition is administered to the putamen of a primate requiring the use thereof. The composition is preferably for use in the treatment of neurodegenerative diseases in primates.

[0016] In all of these embodiments, the transgene encodes a secreted lysosomal protein, preferably human programurin (PGRN), its active fragment, and / or its active variant. Additional embodiments and models of the present invention are disclosed below.

[0017] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the subject matter herein pertains. The following definitions, used herein and in the claims, are provided to facilitate the understanding of the invention. - In this specification, the term "and / or" as used in expressions such as "A and / or B" is intended to include "A and B", "A or B", and "A" and "B". - The singular forms "a," "an," and "the" refer to multiple objects unless otherwise clearly indicated. Therefore, for example, a reference to "recombinant AAV particles" includes "multiple recombinant AAV particles," etc. - The term "contains" does not exclude other elements. For the purposes of this disclosure, the term "consisting of" is considered a preferred embodiment of the term "contains". - The term “viral particle” refers to an infectious, typically replication-deficient viral particle, comprising (i)(ii) at least a portion of a viral vector packaged within a capsid, and optionally (iii) a lipid envelope surrounding the capsid. The term “viral particle” also includes recombinant adeno-associated virus particles. - The terms "recombinant adeno-associated virus particles," "recombinant AAV particles," "rAAV particles," or alternatively, simply "particles," encompass viral particles containing the AAV capsid, which includes the capsid proteins VP1, VP2, and VP3. Differences in the capsid protein sequences of various AAV serotypes result in the use of different cell surface receptors for cell entry. Combined with alternative intracellular processing pathways, each AAV serotype exhibits different tissue tropisms.

[0018] Heterogenetic nucleic acids include a transgene to be expressed in target cells, at least one regulatory sequence that drives the expression of the transgene, and an inverse terminal repeat (ITR) from the viral vector. Aside from the ITR, heterogenetic nucleic acids are primarily composed of non-AAV genomic sequences. - The terms “AAV true type,” “AAVTT,” “AAV-TT,” or “AAVtt” are all defined by reference in International Publication No. 2015121501 and Tordo J. et al., 2018, and relate to capsids containing or consisting of Sequence ID No. 1. The “r” preceding any of these terms indicates recombinant. - The terms "inverted end repeat sequence," "inverted end repeat," or "ITR" refer to sequences located at the 5' and 3' ends of heterologous nucleic acids, which package the transgene and one or more regulatory sequences located between the ITRs within the rAAV capsid. Typically, full-length ITRs from the same source that provides the rep function are used, but alternatively, ITRs from different AAV sources may be used, and cleaved ITRs may also be used as long as they are functional. - The term “regulatory sequence” refers to one or more sequences that direct and / or participate in the expression of a gene (in this specification, a transgene). Typically, the one or more regulatory sequences are selected to drive, assist, and / or control the expression of a transgene in a target tissue, such as the central nervous system (CNS). - The term "transgene" refers to a nucleic acid sequence (typically encoding a protein) that, when administered to primates via rAAV particles according to the present invention, is expressed in the primates, and such sequence is not of AAV origin. Typically, it is of the same origin as the primates treated with the rAAV particles. The term "transgene" should be interpreted as including one or more transgenes. - The term “heterogeneic nucleic acid” refers to nucleic acid sequences packaged within the rAAV capsid that forms the viral particle. Such nucleic acid sequences include the AAV reverse terminal repeat (ITR). In the examples herein, the heterogeneic nucleic acid includes, at least, from 5' to 3', the AAV 5'ITR, (a) a sequence(s) encoding a transgene (i.e., a gene different from the gene encoding the viral protein), and the AAV 3'ITR. - The terms “therapeutic dose” or “therapeutic effective dose” typically refer to the amount or dosage of a compound that, when administered to a primate, exhibits a positive pharmacological and / or physiological effect on the disease and is therefore sufficient to treat the disease. - Terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the onset or worsening of a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or side effects caused by the disease. Thus, treatment covers any treatment of disease in primates, especially humans, and includes (a) preventing the onset of disease in subjects who may be predisposed to the disease but have not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., preventing the onset of the disease, and (c) alleviating the disease, i.e., causing disease regression. [Brief explanation of the drawing]

[0019] [Figure 1] This is a diagram showing coronal sections of the NHP hemisphere stained for the expression of the transgene (PRGN in this specification). A) and B) are respectively the intracapsular administration of vehicle and rAAV-TT. C) shows the staining of the intracerebroventricular administration of rAVV-TT.

Embodiments for Carrying out the Invention

[0020] The present invention is based on the surprising finding that recombinant adeno-associated virus (rAAV) particles containing an AAV wild-type (AAVTT) capsid can diffuse from the capsule into the central nervous system (CNS) when administered intracapsularly and express the transgene encoding progranulin contained in said particles throughout the CNS of primates requiring treatment (particularly the cortex and subcortical regions). The inventors also unexpectedly found that intracapsular administration of rAAV particles according to the present invention is superior to intracerebroventricular (ICV) administration of the same rAAV particles in said primates (Figure 1).

[0021] Therefore, the present invention provides a method for treating neurodegenerative diseases in primates by administering a therapeutic amount of recombinant adeno-associated virus (rAAV) particles to a primate, wherein the rAAV particles a) comprise or consist of an AAV wild-type (AAVTT) capsid containing SEQ ID NO: 1, and b) a heterologous nucleic acid packaged therein and the heterologous nucleic acid i) a 5' inverted terminal repeat (ITR) or a fragment thereof and a 3' ITR or a fragment thereof; ii) a transgene; and iii) one or more regulatory sequences directing the expression of said transgene and the treatment comprises administering said rAAV particles intracapsularly to said primate, and the transgene encodes a secreted lysosomal protein, preferably human progranulin (PGRN), an active fragment thereof and / or an active variant thereof.

[0022] ​ A second object of the present invention is a method for delivering a therapeutic dose of recombinant adeno-associated virus (rAAV) particles to the central nervous system (CNS) of a primate, wherein the rAAV particles are a) A true AAV capsid containing or comprising SEQ ID NO: 1, and b) heterologous nucleic acids packaged within It includes heterogeneous nucleic acids, i) 5' reverse terminal repeat (ITR) or fragment thereof and 3' ITR or fragment thereof; ii) Transgenes; and iii) One or more regulatory sequences that direct the expression of the transgene. Includes, The method comprises the step of administering rAAV particles to the putamen of the primate, wherein the introduced gene encodes a secreted lysosomal protein, preferably human programmeulin (PGRN), its active fragment and / or active variant.

[0023] A third object of the present invention is recombinant adeno-associated virus (rAAV) particles for use in the treatment of neurodegenerative diseases in primates, wherein the rAAV particles are a) A true AAV (AAVTT) capsid containing or comprising SEQ ID NO: 1, and b) heterologous nucleic acids packaged within It includes heterogeneous nucleic acids, i) 5' reverse terminal repeat (ITR) or fragment thereof and 3' ITR or fragment thereof; ii) Transgenes; and iii) One or more regulatory sequences that direct the expression of the transgene. Includes, The rAAV particles are administered to the putamen of the primate, and the transgene encodes a secreted lysosomal protein, preferably human programmeulin (PGRN), its active fragment, and / or its active variant.

[0024] A fourth object of the present invention is recombinant adeno-associated virus (rAAV) particles for use in the delivery of heterologous nucleic acids to the central nervous system of primates, wherein the rAAV particles are a) A true AAV (AAVTT) capsid containing or comprising SEQ ID NO: 1, and b) heterologous nucleic acids packaged within It includes heterogeneous nucleic acids, i) 5' reverse terminal repeat (ITR) or fragment thereof and 3' ITR or fragment thereof; ii) Transgenes; and iii) One or more regulatory sequences that direct the expression of the transgene. Includes, The rAAV particles are administered to the putamen of the primate, and the transgene encodes a secreted lysosomal protein, preferably human programmeulin (PGRN), its active fragment, and / or its active variant.

[0025] A fifth object of the present invention is a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles and one or more carriers and / or excipients, wherein the rAAV particles are a) A true AAV capsid containing or comprising SEQ ID NO: 1, and b) heterologous nucleic acids packaged within It includes heterogeneous nucleic acids, i. 5' reverse terminal repeat (ITR) or fragment thereof and 3' ITR or fragment thereof; ii. Transgenes; and iii. One or more regulatory sequences that direct the expression of the transgene. Includes, The pharmaceutical composition is administered to the putamen of a primate in need, and the transgene encodes a secreted lysosomal protein, preferably human progranulin (PGRN), its active fragment, and / or its active variant. The composition is preferably used for the treatment of neurodegenerative diseases in primates.

[0026] In the overall context of the present invention, when administered to the putamen, the particles can diffuse into the central nervous system (CNS) and thus be delivered to the entire region of the CNS, as indicated by the expression of the transgene throughout the CNS.

[0027] In the overall context of the present invention, a 5'ITR includes or comprises i) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2; or ii) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5; and a 3'ITR includes or comprises i) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3; or ii) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6. In a non-limiting example, a 5'ITR includes or comprises SEQ ID NO: 2; and a 3'ITR includes or comprises SEQ ID NO: 3. In another non-restrictive example, 5'ITR contains or consists of sequence number 5, and 3'ITR contains or consists of sequence number 6.

[0028] In the overall context of the present invention, heterologous nucleic acids include transgenes encoding secretory lysosomal proteins (including pre- and / or proforms of such secretory proteins). The secretory lysosomal proteins are preferably human programurin (PGRN), its active fragment and / or active variant, and the PGRN preferably has an amino acid sequence comprising SEQ ID NO: 4 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. Non-limiting examples of nucleic acid sequences encoding PGRN include SEQ ID NO: 7 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0029] The heterologous nucleic acid according to the present invention comprises one or more regulatory sequences that direct, assist and / or control the expression of the transgene in primates treated by gene therapy with rAAV particles as described herein. In the overall context of the present invention, the one or more regulatory sequences that direct the expression of the transgene are: a. One or more transcription start sequences (such as promoters), b. One or more translation initiation sequences, c. One or more mRNA stability sequences, d. One or more polyadenylated sequences, e. One or more secretory sequences, f. One or more enhancer sequences, g. One or more introns, h. One or more TATA boxes, i. One or more microRNA target sequences, j. One or more polylinker sequences that facilitate the insertion of DNA fragments into the vector. k. One or more splicing signal sequences, l. One or more transcription termination sequences (such as polyadenylated sequences) or m. One or more combinations of groups a through l. It is selected from the group consisting of the following.

[0030] As used herein, the term “promoter” refers to a regulatory element that directs the transcription of an activatably ligated transgene. Promoters can regulate both the rate and efficiency of transcription of an activatably ligated transgene. Promoters can also be activatably ligated to other regulatory elements that enhance (“enhancer”) or repress (“repressor”) the promoter-dependent transcription of the transgene. These regulatory elements include, but are not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from a promoter, such as atenuators, enhancers and silencers. Promoters are generally located near the transcription start site of an activatably ligated transgene, on the same strand, and upstream of the DNA sequence (towards the 5' region of the sense strand).

[0031] As used herein, the term “operably linked” refers to the linking of elements that are functionally related. A transgene is “operably linked” when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or transcriptional regulatory sequence is operably linked to a transgene if it affects the transcription of the transgene. Being operably linked means that the linked DNA sequences are typically contiguous.

[0032] Preferably, one or more regulatory sequences according to the present invention contained in a heteronucleotide are specifically selected to drive the expression of the transgene in the central nervous system (CNS). In one preferred embodiment, the one or more regulatory sequences include a neural promoter. Thus, the heteronucleotide construct may include any regulatory elements listed above, either alone or in any combination of two or more elements, any combination of three or more elements, any combination of four or more elements, any combination of five or more elements, and so on. In another embodiment, the one or more regulatory sequences include or consist of a promoter and a transcription termination sequence. In a further embodiment, the one or more regulatory sequences include or consist of a promoter, an enhancer (such as one or more introns), and a polyadenylation site. Generally, the heteronucleotide includes regulatory sequences that precede (5' non-coding sequence) and follow (3' non-coding sequence) the coding sequence required for the expression of the transgene. Therefore, in certain embodiments, the heterologous nucleic acid includes at least (i)(ii) a transgene under the control of a promoter, and (iii) a 3' untranslated region comprising a normally polyadenylated sequence / site and / or transcription terminator.

[0033] In another embodiment, the neural promoter includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8. In another embodiment, the neural promoter may be bound to a naturally occurring or manipulated untranslated sequence such as SEQ ID NO: 9, 11, 12, 13, 14, or 16. In an unrestricted example, the sequence combining the neural promoter and the untranslated sequence includes or consists of a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15.

[0034] As used herein, the terms “polyadenylation signal,” “polyadenylation site,” or “poly(A) signal / site” refer to a specific recognition sequence within the 3' untranslated region (3'UTR) of a gene that is transcribed into a precursor mRNA molecule and leads to the termination of gene transcription. The poly(A) signal acts as a signal for endo-nucleic cleavage of the 3' end of the newly formed precursor mRNA and the addition of an RNA stretch consisting solely of adenine bases to this 3' end (the polyadenylation process; poly(A) tail). The poly(A) tail is important for mRNA nuclear export, translation, and stability. In the context of the present invention, a polyadenylation signal is a recognition sequence that can direct the polyadenylation of mammalian genes and / or viral genes in mammalian cells. In further embodiments, the polyadenylation sequence comprises or consists of SEQ ID NO: 17, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0035] A non-limiting example of heterogeneous nucleic acids that can be packaged in rAAV particles is the sequence containing SEQ ID NO: 18.

[0036] Non-limiting embodiments of neurodegenerative diseases according to the present invention include slowly progressive and rapidly progressive neurodegenerative diseases, such as neuronal ceroid lipofuscinosis (NCL11, etc.), mucopolysaccharidosis (MPS), such as MPS IIIC (O'Leary C. et al., 2016), progressive supranuclear palsy (PSP), corticobasal syndrome, Parkinson's disease, Lewy body dementia, Alzheimer's disease, or amyotrophic lateral sclerosis (ALS or ALS-D) with or without dementia.

[0037] In the overall context of the present invention, neurodegenerative diseases are preferably adult-onset neurodegenerative diseases. Examples of preferred adult-onset neurodegenerative diseases that can be treated include frontotemporal dementia (FTD), NCL11 type, and / or PGR haploinsufficiency.

[0038] The appropriate dosage of rAAV particles according to the present invention can be determined by those skilled in the art. The dosage selected depends on various pharmacokinetic factors, including the time of administration, the diffusion rate of the rAAV particles, the expression rate of the transgene, the frequency of administration, the optional presence of other drugs, compounds and / or materials used in combination with the specific rAAV particles, the age, sex, weight, condition, general health and previous medical history of the patient being treated.

[0039] Generally, the preferred total dosage of rAAV particles containing AAVTT capsids to be administered in accordance with the present invention is at least 1×10 6 vg of rAAV particles. Preferably, the preferred total dosage of rAAV particles containing AAVTT capsids to be administered in accordance with the present invention is in the range of about 1×10 6 to about 1×10 20 , in the range of about 1×10 8 to about 1×10 18 , in the range of about 1×10 10 to about 1×10 16 , or in the range of about 1×10 12 to about 1×10 14 vg of rAAV particles.

[0040] In the overall context of the present invention, the total dose of rAAV particles can be administered to primates as a single dose or as multiple doses (e.g., two or more doses, three or more doses, four or more doses, six or more doses, etc.). When multiple doses are administered, they can be administered simultaneously or consecutively. The target may also be one hemisphere's putamen or both hemispheres' putamen. Preferably, the particles are administered to each hemisphere's putamen to enable homogeneous particle distribution and / or homogeneous expression of the transgenes contained in the particles. When particles are administered to two hemispheres, each hemisphere may receive one or more doses. If the putamen of each hemisphere receives more than one dose, the dose is typically administered at different but still intraputation sites within the putamen (two intraputation sites if two doses are administered, three administration sites if three doses are administered, and four doses if four doses are administered) to further improve the homogeneous distribution of the particles and the proteins expressed by the transgenes incorporated into the particles.

[0041] For example, with respect to the total dose of rAAV particles to be administered, two equal doses of rAAV particles can be administered to the putamen of the right and left hemispheres, respectively, to deliver an equal dose equivalent to half of the total dose to each hemisphere. Alternatively, the total dose can be divided among the putamen of each hemisphere as desired.

[0042] In the overall context of this invention, "primate" refers to either a human or a non-human primate. Alternatively, the terms "patient" or "subject" may be used interchangeably instead of "primate."

[0043] The recombinant adeno-associated virus (rAAV) particles according to the present invention may be included in a pharmaceutical composition together with one or more carriers and / or excipients. The term "carrier" includes all solvents, dispersion media, isotonic agents, and absorption retarders, as long as they are physiologically compatible and suitable for administration to the central nervous system of primates in the context of the present invention. Examples of carriers include one or more of water, physiological saline, phosphate-buffered saline, buffers, and combinations thereof.

[0044] The rAAV particles(s) according to the present invention can be generated by conventional methods and protocols. Simply put, the viral particles can be generated in a host cell, more specifically in a specific virus-producing cell (packaging cell), which is transfected with a suitable heterogeneous nucleic acid to be packaged in the presence of a helper vector or virus or other DNA construct(s). As used herein, the term “packaging cell” refers to a cell or cell line that can be transfected with the heterogeneous nucleic acid of this disclosure via a suitable plasmid and that in transfects all the missing functions required for the complete replication and packaging of viral particles.

[0045] Typically, the process for generating viral particles includes a) culturing packaging cells containing the nucleic acid construct or viral vector described above in a culture medium; b) collecting viral particles from the cell culture supernatant and / or from within the cells; c) purifying the viral particles, typically by at least affinity chromatography and / or ion chromatography; and d) optionally formulating the viral particles to obtain a pharmaceutical composition.

[0046] The present invention also comprises a method for delivering a therapeutic dose of heterologous nucleic acid to the central nervous system (CNS) of a primate, wherein the heterologous nucleic acid is packaged in recombinant adeno-associated virus (rAAV) particles comprising a true AAV capsid comprising or consisting of Sequence ID No. 1, the heterologous nucleic acid comprising i) a 5' inverted terminal repeat (ITR) or a fragment thereof and a 3' ITR or a fragment thereof; ii) a transgene; and iii) one or more regulatory sequences that direct the expression of the transgene, the method comprising the step of administering the rAAV particles to the putamen of the primate, the transgene encoding a secreted lysosomal protein, preferably human programmeurin (PGRN), an active fragment thereof and / or an active variant thereof.

[0047] The present invention also comprises a method for expressing a transgene in the central nervous system (CNS) of a primate, wherein the transgene is contained in heterogeneous nucleic acid packaged in recombinant adeno-associated virus (rAAV) particles containing a true AAV capsid containing or comprising Sequence ID No. 1, the heterogeneous nucleic acid further comprising i) a 5' reverse terminal repeat (ITR) or a fragment thereof and a 3' ITR or a fragment thereof; and ii) one or more regulatory sequences that direct the expression of the transgene, the method comprising the step of administering the rAAV particles to the putamen of the primate, further expressing the transgene packaged in the particles for diffusion of the rAAV particles within the CNS, wherein the transgene encodes a secreted lysosomal protein, preferably human programmeurin (PGRN), an active fragment thereof and / or an active variant thereof.

[0048] The present invention also comprises a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles and one or more carriers and / or excipients, wherein the rAAV particles comprise a) an AAV true-type capsid comprising or comprising SEQ ID NO: 1, and b) a heterogeneous nucleic acid packaged therein, the heterogeneous nucleic acid comprising i. a 5' inverted terminal repeat (ITR) or a fragment thereof and a 3' ITR or a fragment thereof; ii. a transgene; and iii. one or more regulatory sequences that direct the expression of the transgene; the pharmaceutical composition is formulated for intramucosal administration to primates requiring it, wherein the transgene encodes a secreted lysosomal protein, preferably human progranulin (PGRN), an active fragment thereof and / or an active variant thereof, comprising the pharmaceutical composition. The composition is preferably for use in the treatment of neurodegenerative diseases in primates.

[0049] Array description [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table A-7] [Table A-8] [Examples]

[0050] material rAAV particles containing AAVtt were used. The heterologous nucleic acid (SEQ ID NO: 18) packaged in these particles contained the granulin (PGRN) gene as an introduced gene. The particles were generated using a routine triple transfection method for mammalian cell systems.

[0051] This study used 24 male cynomolgus monkeys (2 years of age or older, of Mauritius origin). The particle stock was provided in 25 μL aliquots, thawed, combined as needed (depending on the total volume administered), and dispensed at each dose. Each aliquot was prepared and used on the day of administration.

[0052] method Intraventricular (ICV) administration Intraventricular (ICV) administration was performed as follows: Cerebrospinal fluid (CSF) was collected as a control / baseline before the start of the study. Similarly, magnetic resonance imaging (MRI) was performed before the start of the study. At the start of the study, an incision was made, the skin was inverted, and a single hole was drilled in the skull above the target location (left hemisphere lateral ventricle). A 22-gauge needle was used. The position of the needle inside the ventricle was confirmed by contrast injection and fluoroscopy. The test material was then administered into the lateral ventricle at a rate of 0.1 mL / min for a total administration time corresponding to the volume. The injection needle was left in place for 2 minutes after the completion of administration, and then withdrawn. After the completion of administration, the skin was closed in a standard manner, and the animals were allowed to recover. The animals were monitored for 62 days postoperatively.

[0053] Intramucosal administration Intraputaminal administration was performed as follows: Magnetic resonance imaging (MRI) was performed before the start of the study as a control / baseline. An incision was made and the skin was inverted at the start of the study. A hole was drilled in the skull at the target site. The syringe was primed three times to ensure that the barrel could move freely. Administration was started at 1 μL / min as soon as the device was lowered into place (approximately 1 minute to the target depth).

[0054] After reaching the predetermined depth, the test material was administered at a rate of 2 μL / min for 5 minutes, then increased to a rate of 1 μL / min until it reached 5 μL / min. The flow rate was then maintained at 5 μL / min for the remainder of the administration time. The cannula was left in place for 10 minutes after the end of administration, then withdrawn and removed. The skin was closed using standard methods, and the animals were allowed to recover. The animals were monitored for 62 days post-operatively.

[0055] Histological and microscopic evaluation Materials for histological and microscopic evaluation were fixed by immersion in methanol-free 4% paraformaldehyde (PFA) in 1× phosphate-buffered saline (PBS) at room temperature for 72–96 hours, then transferred to 1× PBS and kept at 2–8°C until use. Representative slides were stained for PGRN (monkey PGRN only (see Figure 1A), or both human PGRN and monkey PGRN (see Figures 1B and 1C)) using standard protocols and techniques.

[0056] (Example 1) In vivo distribution study in non-human primates The purpose of this study is to evaluate the in vivo distribution of AAVTT-mediated gene therapy products when administered via intraventricular (ICV) or intraputaminal routes in cynomolgus monkeys, and to investigate the suitability of various administration route and dosage combinations for reaching an appropriate cerebral range for indications of neurodegenerative diseases.

[0057] Animals were administered a test substance selected from AAV-TT-based particles containing progranulin as the transgene, or from a vehicle (PBS containing 0.001% Pluronic® F68). The following administration methods were used: intraventricular administration (ICV) - unilateral administration (Group 1) and intraparenchymal administration (IP) - pre- and post-compression, bilateral administration (Groups 2 and 3).

[0058] The experimental design can be found in Table 1. [Table 1]

[0059] As shown in Figure 1B, when particles were administered at a dose of approximately 7.9 e12 vg, a clear and extensive distribution of PGRNs across cortical and subcortical regions was made possible by intraputamen administration. In contrast, no staining was observed with the vehicle (Figure 1A). As shown in Figure 1C, only minimal expression of PGRNs was observed with intraventricular administration at a dose of 1 e13 vg (a higher dose than that via the intraputamen pathway). The only exception was the location of the pathway (indicated by the arrow in Figure 1C).

[0060] These results surprisingly showed that recombinant adeno-associated virus (rAAV) particles containing the true AAV (AAVTT) capsid, when administered to the putamen, could diffuse from the putamen into the central nervous system (CNS) and express the PGRN-transgene contained in the particles throughout the CNS (particularly the cortical and subcortical regions) of primates requiring treatment. In particular, administration of rAAV particles to the putamen according to the present invention was superior to intraventricular (ICV) administration of the same rAAV particles in the primates (Figure 1). These results support recombinant adeno-associated virus (rAAV) particles for use in the treatment of neurodegenerative diseases in primates (non-human primates and humans), wherein the rAAV particles comprise a) an AAV true type (AAVTT) capsid comprising or comprising SEQ ID NO: 1, and b) heterogeneous nucleic acids packaged therein, the heterogeneous nucleic acids comprising i) a 5' inverted terminal repeat (ITR) or fragment thereof and a 3' ITR or fragment thereof; ii) a transgene; and iii) one or more regulatory sequences directing the expression of the transgene, the rAAV particles being administered to the putamen of the primate, and the transgene encoding a secretory lysosomal protein, preferably human programmeurin (PGRN), its active fragment and / or active variant.

[0061] References Ojala DS et al., 2015, The Neuroscientis, 21(1): 84-98 Bartlett JS et al., 1998, Hum. Gene Ther., 9(8):1181-6 Mandel RJ et al., 2004, Curr. Opin. Mol. Ther.: 6(5):482-90 Wood et al. 2022, Front Mol Biosci, 9:965089 Coyle-Gilchrist IT et al., 2016, Neurology, 86(18):1736-43 Rohrer JD. et al., 2009, Neurology, 73(18):1461-6 Le Ber I., et al., 2008, Brain, 131(3):732-46 Kamate M. et al., 2019, Brain, 41(6):542-545 Irwin D. et al., 2009, J. Neurol. Sci., 276(1-2): 9-13 Wozar F. et al., 2022, Klin. Mobl. Augenheilkd., 239(3):270-74 Pignataro D. et al., 2018, J. Neural. Transm., 125:575-589 FDA draft guidance for industry “Human Gene Therapy for Neurodegenerative Diseases”, 2022 WO2015121501 Tordo J. et al., 2018, Brain, 141:2014-31 O’Leary C. et al., 2016, Molecular Genetics and Metabolism, 120:S254

Claims

1. A method for treating neurodegenerative diseases in primates by administering a therapeutic dose of recombinant adeno-associated virus (rAAV) particles to primates, wherein the rAAV particles are a) A true AAV (AAVTT) capsid containing or comprising Sequence ID No. 1, and b) Heterogeneous nucleic acids packaged within It includes heterogeneous nucleic acids, i) 5' reverse terminal repeats (ITRs) or fragments thereof, and 3' ITRs or fragments thereof; ii) Transgenes; and iii) One or more regulatory sequences that direct, assist, and / or control the expression of the introduced gene. Includes, The procedure comprises administering the rAAV particles to the putamen of the mammal, wherein the transgene encodes a secretory lysosomal protein, preferably human progranulin (PGRN), its active fragment and / or active variant.

2. A method for delivering a therapeutic dose of recombinant adeno-associated virus (rAAV) particles to the central nervous system of a primate, wherein the rAAV particles are a) A true AAV capsid containing or comprising Sequence ID No. 1, and b) Heterogeneous nucleic acids packaged within It includes heterogeneous nucleic acids, i) 5' reverse terminal repeats (ITRs) or fragments thereof, and 3' ITRs or fragments thereof; ii) Transgenes; and iii) One or more regulatory sequences that direct, assist, and / or control the expression of the introduced gene. Includes, The method comprises the step of delivering rAAV particles to the putamen of the primate, wherein the introduced gene encodes a secreted lysosomal protein, preferably human programrunulin (PGRN), an active fragment thereof, and / or an active variant thereof.

3. Recombinant adeno-associated virus (rAAV) particles for use in the treatment of neurodegenerative diseases in primates, wherein the rAAV particles are a) A true AAV (AAVTT) capsid containing or comprising Sequence ID No. 1, and b) Heterogeneous nucleic acids packaged within It includes heterogeneous nucleic acids, i) 5' reverse terminal repeats (ITRs) or fragments thereof, and 3' ITRs or fragments thereof; ii) Transgenes; and iii) One or more regulatory sequences that direct, assist, and / or control the expression of the introduced gene. Includes, The rAAV particles are administered to the putamen of the primate, and the transgene encodes a secretory lysosomal protein, preferably human progranulin (PGRN), its active fragment and / or active variant.

4. Recombinant adeno-associated virus (rAAV) particles for use in delivering heterologous nucleic acids to the central nervous system of primates, wherein the rAAV particles are a) A true AAV (AAVTT) capsid containing or comprising Sequence ID No. 1, and b) Heterogeneous nucleic acids packaged within It includes heterogeneous nucleic acids, i) 5' reverse terminal repeats (ITRs) or fragments thereof, and 3' ITRs or fragments thereof; ii) Transgenes; and iii) One or more regulatory sequences that direct, assist, and / or control the expression of the introduced gene. Includes, The rAAV particles are administered to the putamen of the primate, and the transgene encodes a secretory lysosomal protein, preferably human progranulin (PGRN), its active fragment and / or active variant.

5. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles and one or more carriers and / or excipients, wherein the rAAV particles are a) A true AAV capsid containing or comprising Sequence ID No. 1, and b) Heterogeneous nucleic acids packaged within It includes heterogeneous nucleic acids, i. 5' reverse terminal repeats (ITRs) or fragments thereof, and 3' ITRs or fragments thereof; ii. Transgenes; and iii. One or more regulatory sequences that direct, assist, and / or control the expression of the introduced gene. Includes, The pharmaceutical composition is administered to the putamen of a primate in need thereof, and the transgene encodes a secretory lysosomal protein, preferably human progranulin (PGRN), its active fragment and / or its active variant.

6. The pharmaceutical composition according to claim 5, wherein the composition is for use in the treatment of neurodegenerative diseases in primates.

7. A method according to any one of claims 1 to 6, rAAV particles or pharmaceutical compositions for use therewith, wherein human programmeulin (PGRN), its active fragment and / or its active variant, is encoded by 1) an amino acid sequence comprising SEQ ID NO: 4, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith, or 2) a nucleic acid sequence encoding PGRN comprising SEQ ID NO: 7, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith.

8. The method according to any one of claims 1 to 7, rAAV particles or pharmaceutical composition for use, wherein the neurodegenerative disease is neuronal ceroid lipofuscinosis, for example, NCL11 type, mucopolysaccharidosis (MPS), progressive supranuclear palsy (PSP), corticobasal syndrome, Parkinson's disease, Lewy body dementia, Alzheimer's disease, or amyotrophic lateral sclerosis (ALS or ALS-D) with or without dementia.

9. The method according to any one of claims 1 to 7, rAAV particles or pharmaceutical composition for use, wherein the neurodegenerative disease is an adult-onset neurodegenerative disease.

10. The method according to claim 9, rAAV particles or pharmaceutical composition for use, wherein the disease is frontotemporal dementia (FTD), NCL11 type and / or PGRN haploinsufficiency.

11. The method according to any one of claims 1 to 10, or rAAV particles or a pharmaceutical composition for use, wherein rAAV particles containing an AAVTT capsid diffuse from the putamen to the cortex and subcortical regions.

12. The administration is at least 1 × 10 ６ A method according to any one of claims 1 to 11, or rAAV particles or a pharmaceutical composition for use, comprising a total dose containing rAAV particles of a viral genome (vg).

13. The method according to any one of claims 1 to 12, rAAV particles or pharmaceutical composition for use, wherein administration of rAAV particles containing an AAVTT capsid into the putamen is superior to intraventricular (ICV) administration of rAAV particles in the primate.

14. The method, rAAV particles, or pharmaceutical composition for use according to any one of claims 1 to 13, wherein the total dose administered comprises at least two doses, preferably one dose administered to the putamen of the right hemisphere and one dose administered to the putamen of the left hemisphere.

15. a) 5'ITR is, i) Sequence ID 2, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; or ii) Sequence ID 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. It includes or consists of; b) 3'ITR is, i) Sequence ID 3, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; or ii) Sequence ID 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. Includes or consists of A method according to any one of claims 1 to 14, or rAAV particles or a pharmaceutical composition for use.

16. One or more regulatory sequences that direct, assist, and / or control the expression of the aforementioned transgene, a. One or more transcription start sequences (such as promoters), b. One or more translation initiation sequences, c. One or more mRNA stability sequences, d. One or more polyadenylated sequences, e. One or more secretory sequences, f. One or more enhancer sequences, g. One or more introns, h. One or more TATA boxes, i. One or more microRNA target sequences, j. One or more polylinker sequences that promote the insertion of DNA fragments into the vector, k. One or more splicing signal sequences, l. One or more transcription termination sequences (such as polyadenylated sequences) or m. One or more combinations of groups a. through l. A method according to any one of claims 1 to 15, rAAV particles or pharmaceutical compositions for use, selected from the group consisting of the above.

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