Capsid and neuronal targeting methods

Abstract

AAV capsids and methods useful for transducing neurons, particularly both upper and lower motor neurons, are provided herein. In some embodiments, these methods and compositions are useful for delivering nucleic acid molecules to neurons. In one example, a targeted sequence is used to guide the capsid to the nucleic acid in the delivery of the nucleic acid molecule.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 499,457, filed on 1 May 2023, which is incorporated herein by reference. [Background technology]

[0002] Motor neuron diseases (MNDs) are a group of neurological disorders that destroy motor neurons, which are cells crucial for controlling skeletal muscle function for activities such as walking, breathing, speaking, and swallowing. Generally, messages or signals from upper motor neurons in the brain are typically transmitted to the brainstem and spinal cord, and then to the muscles in the body via lower motor neurons. For example, upper motor neurons instruct lower motor neurons to produce muscle movement. If muscles cannot receive signals from motor neurons, the muscles begin to weaken and shrink in size (e.g., muscle atrophy or wasting). Muscles may also exhibit spontaneous twitching (fasciculation) and / or stiffness (spasticity), as well as hyperactive reflexes that make voluntary movement slow and difficult. Over time, individuals with MND may lose the ability to walk or control other movements. [Overview of the project]

[0003] Recombinant adeno-associated virus (AAV) vector-mediated gene therapy is one of the most promising approaches for improving the genetic morphology of neuromuscular diseases. However, while AAV9 is clinically used to treat motor neuron disorders, wild-type AAV9 requires high vector doses (>10) to deliver therapeutic effects. 14The use of a vector genome (vg / kg) is still required. Furthermore, AAV9 effectively transduces a wide range of cell types, resulting in substantial vector spillover to non-target cells in the body. Such high dose requirements lead to several significant problems, including off-target toxicity, a limited therapeutic window, and high manufacturing costs.

[0004] Variant AAVs containing the variant capsid protein (e.g., variant capsid) described herein are useful for transducing motor neurons. For example, AAVs containing the variant capsid protein (e.g., variant capsid) described herein exhibit enhanced motor neuron tropism.

[0005] In some embodiments, methods for delivering nucleic acid molecules to motor neurons of an individual are provided herein, the methods comprising the steps of administering a variant adeno-associated virus (AAV) comprising a nucleic acid molecule to the individual, wherein the variant AAV comprises a variant capsid protein having an amino acid insertion in loop VIII, and the amino acid insertion comprises SEQ ID NO: 15.

[0006] In some embodiments, methods for delivering therapeutic nucleic acid molecules to motor neurons of subjects having motor neuron disease are provided herein, the methods comprising the step of administering a variant adeno-associated virus (AAV) containing a therapeutic nucleic acid molecule to an individual, wherein the variant AAV contains a variant capsid protein having an amino acid insertion in loop VIII, and the amino acid insertion has the sequence number 15.

[0007] In some embodiments, methods are provided herein for treating a disease or disorder of motor neurons in an individual, the method comprising the step of administering a variant adeno-associated virus (AAV) comprising a therapeutic nucleic acid molecule, wherein the variant AAV comprises a variant capsid protein having an amino acid insertion within loop VIII, and the amino acid insertion comprises SEQ ID NO: 15. Motor neuron disorders generally refer to and encompass diseases that interfere with the normal function of motor neurons. In some embodiments, motor neuron disorders are characterized by muscle atrophy or wasting and / or fasciculations.

[0008] In some embodiments, methods for delivering nucleic acids to motor neurons are provided herein, the methods comprising the step of contacting a motor neuron with a variant adeno-associated virus (AAV) comprising a nucleic acid molecule, wherein the variant AAV comprises a variant capsid protein having an amino acid insertion in loop VIII, and the amino acid insertion comprises SEQ ID NO: 15.

[0009] In some embodiments, the motor neurons are upper motor neurons. In some embodiments, the motor neurons are lower motor neurons. In some embodiments, the motor neurons include both upper and lower motor neurons. In some embodiments, the motor neurons belong to an individual. In some embodiments, the motor neurons are located within the frontal cortex of the brain. In some embodiments, the motor neurons are located within the spine. In some embodiments, the motor neurons are located within the anterior horn region of the spinal cord. In some embodiments, the motor neurons are located within the cervical to sacral region of the spine. In some embodiments, the motor neurons are located within the cervical region of the spine, the thoracic region of the spine, the lumbar region of the spine, or the sacral region of the spine.

[0010] In some embodiments, the variant capsid protein is a variant AAV serotype 9 (AAV9) capsid protein. In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertion includes SEQ ID NO: 9.

[0011] In some embodiments, the AAV9 capsid protein contains the N272A mutation. In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertion includes SEQ ID NO: 14.

[0012] In some embodiments, the amino acid insertion further includes a linker. In some embodiments, the amino acid insertion is formula L1-X-L2 (In the formula, L1 contains the first amino acid linker sequence, X includes sequence number 15, L2 is the second linker sequence. It also includes.

[0013] In some embodiments, L1 and L2 are different sequences. In some embodiments, L1 and L2 are the same sequence.

[0014] In some embodiments, the variant capsid includes SEQ ID NO: 15, and the remaining capsid sequence has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16. [Brief explanation of the drawing]

[0015] Novel features of the variant AAV described herein are specifically described in the appended claims. A better understanding of the features and advantages of the variant AAV described herein can be obtained by referring to the following detailed description and appended drawings which describe exemplary embodiments in which the principles of the present invention are utilized.

[0016] [Figure 1] A diagram of the TRAnscription-dependent Directed Evolution (TRADE) construct used for the discovery of variant capsids is shown. [Figure 2] This paper presents data demonstrating that AAV-CAP2 exhibits broad central nervous system (CNS) transduction in cynomolgus macaques. (A) shows quantification of neuronal transduction efficiency in different brain regions 3 weeks after intravenous injection of AAV-CAP2-CAG-GFP. (B) shows representative confocal images of cynomolgus macaque brain sections immunostained with anti-GFP (green) and anti-NeuN (red) antibodies across various CNS regions of interest. Yellow / orange cells represent co-expression of GFP and NeuN. Scale bars are 100 μm. (C) shows quantification of neuronal specificity of AAV-CAP2-transduced cells in different brain regions. Four to six brain sections were stained, imaged, and technical replicas were pooled to obtain single neuronal transduction efficiency or marker overlap for each region. Error bars are expressed as standard deviation. [Figure 3]Data are shown demonstrating that AAV-CAP2 targets upper motor neurons / Betz cells in the motor cortex of cynomolgus monkeys. (A) shows a representative image of DAB-GFP labeling in the primary motor cortex. (B) shows the cell size distribution of GFP-positive transfected cells in layer Vb of the motor cortex of cynomolgus monkeys. It is a frequency histogram showing the number of motor neurons (binned in 100-μm2 steps) and the size of motor neurons (μm2) in each size bin. (C) shows a representative image of double immunofluorescence microscopy for SMI-32 (red) and GFP (green). The scale bar is 50 μm. (D) shows the quantification of upper motor neuron transduction efficiency. (E) Specificity of upper motor neurons among all vector-transfected neurons. Four to six brain sections from the dorsal part of Brodmann area 4 of the motor cortex were stained, imaged for each animal, and technical replicates were pooled to obtain the mean for each animal. Data are presented as mean + / − standard deviation. [Figure 4] Data are shown demonstrating the in vivo distribution of the AAV-CAP2-CAG-GFP vector genome in the brain of cynomolgus monkeys after systemic delivery. (A) shows the quantification of vector genome copy number in cortical and putamen samples by droplet digital PCR (ddPCR). (B) shows a table comparing the results with the vector genome copy number in the non-human primate (NHP) motor cortex reported in the literature. [Figure 5] Data are shown demonstrating that intravenous AAV-CAP2 delivery mediates robust gene expression in the spinal cord. (A) shows a schematic diagram of a transverse (left) and longitudinal coronal (right) section of the spinal cord. (B) shows representative DAB-GFP immunohistochemistry in coronal (B–D) and transverse (E–I) sections of the cervical (B, E, F), thoracic (C, G), and lumbar (D, H, I) segments of the spinal cord. The DAB-GFP signal was detected in cells of the anterior horn with a motor neuron-like morphology. (E, I) show enlarged views of the native GFP fluorescence signal in anterior horn cells in the cervical and lumbar segments (dotted lines), respectively. [Figure 6]Data are shown demonstrating that AAV-CAP2 exhibits tropism for lower motor neurons after systemic delivery in cynomolgus monkeys. (A–B) show representative sections of the cervical (A) and lumbar (B) ventral horns from cynomolgus monkey spinal cord observed by laser scanning confocal microscopy 21 days after i.v. injection. Co-localization of ChAT-immunolabeled motor neurons (MN, red) and GFP (green) was observed in the ventral horn. (C) shows quantification of lower motor neuron transduction in the cervical and lumbar regions. Data are presented as mean + / − standard deviation. [Figure 7] Data are shown demonstrating tropism in the thoracic and lumbar spinal cord. (A) shows the vector genome copy number per diploid genome detected in the spinal cord of two cynomolgus monkeys 3 weeks after i.v. injection of AAV-CAP2-CAG-GFP. (B) shows a table comparing the results to vector genome copy numbers in NHP spinal cord reported in the literature. [Figure 8] Data are shown demonstrating DRG transduction by AAV-CAP2 in cynomolgus monkeys after i.v. injection. (A) shows a representative lumbar DRG section stained 3 weeks after i.v. injection of AAV-CAP2-CAG-GFP. Each section was immunostained with antibodies against GFP (green) and NeuN (red). Yellow / orange cells represent co-expression of GFP and NeuN. (B) shows quantification of lumbar DRG sections expressing GFP. All data are presented as mean + / − standard deviation. (C) shows the results of vector genome copy number assessment for the lumbar DRG of cynomolgus monkeys. [Figure 9] Data are shown demonstrating biodistribution in peripheral organs. (A) The vector genome copy number per diploid genome in the liver and heart of two cynomolgus monkeys was determined 3 weeks after i.v. injection of AAV-CAP2-CAG-GFP. (B) shows a table comparing the results to vector genome copy numbers in NHP liver reported in the literature. [Figure 10]This document presents data demonstrating the transduction and specificity of AAV-CAG-CAP2-GFP neurons in the mouse CNS. (A) shows the quantification of GFP+ cells within NeuN+ cells in the motor cortex, striatum, and thalamus. (B) shows the quantification of total NeuN+ cells within GFP+ cells in the motor cortex, striatum, and thalamus. N=5 mice / group. Data are presented as mean + / - standard deviation. [Figure 11] This document presents data demonstrating AAV-CAP2 transduction in the mouse motor cortex. (A-B) shows representative images from layer Vb of the mouse primary motor cortex stained 3 weeks after IV injection of AAV-CAP2-CAG-GFP. Yellow arrows: GFP+ cells with corticospinal upper motor neuron morphology. White arrows indicate GFP+ pyramidal cells with corticospinal morphology. Representative images of the mouse motor cortex immunostained with anti-GFP (green), anti-NeuN (blue), and anti-SMI-32 (red) antibodies. Yellow arrows indicate SMI-32+ / GFP+ corticospinal upper motor neurons. (C) shows quantification of upper motor neuron transduction in mice. (D) The figure shows the specificity of upper motor neurons in all vector-transduced neurons (n=5 mice). Data are presented as mean + / - standard deviation. [Figure 12] This paper presents data demonstrating AAV-CAP2 transduction in mouse spinal cord. (A) shows a representative section of the lumbar anterior horn from mouse spinal cord, observed by laser scanning confocal microscopy 21 days after IV injection. White arrows indicate the colocalization of ChAT immunolabeled motor neurons (MNs) with GFP and NeuN observed in the anterior horn. (B) shows quantification of transduction efficiency of panneurons and motor neurons in the cervical and lumbar segments of mouse spinal cord (n=5 mice). Data are presented as mean + / - standard deviation. [Figure 13]This paper presents data demonstrating AAV-CAP2-mediated brain transduction in mice and cynomolgus monkeys. (A) shows the neuronal transduction efficiency in the motor cortex, striatum, and thalamus of mice (n=5) and cynomolgus monkeys (n=2). (B) shows the upper motor neuron transduction efficiency in mice (n=5) and cynomolgus monkeys (n=2). Data are presented as mean + / - standard deviation. [Figure 14A] Figures 14A-14G show the amino acid alignment of the AAV serotype. [Figure 14B] Figures 14A-14G show the amino acid alignment of the AAV serotype. [Figure 14C] Figures 14A-14G show the amino acid alignment of the AAV serotype. [Figure 14D] Figures 14A-14G show the amino acid alignment of the AAV serotype. [Figure 14E] Figures 14A-14G show the amino acid alignment of the AAV serotype. [Figure 14F] Figures 14A-14G show the amino acid alignment of the AAV serotype. [Figure 14G] Figures 14A-14G show the amino acid alignment of the AAV serotype. [Modes for carrying out the invention]

[0017] This specification provides variant adeno-associated virus (AAV) capsids useful for targeting motor neurons. For example, variant AAV includes the described variant capsids useful for delivering nucleic acid molecules to motor neurons. Generally, the variant capsids described herein utilize amino acid insertions (e.g., SEQ ID NO: 15) that confer efficient and specific targeting (e.g., transduction) of motor neurons. The efficient and specific targeting of motor neurons achieved by these variant capsids and their methods of use offers advantages over conventional wild-type capsid sequences used to deliver nucleic acids to motor neurons (e.g., wild-type AAV9 of SEQ ID NO: 9).

[0018] Variant capsids for introducing motor neurons Variant AAVs containing a variant capsid protein with an amino acid insertion including SEQ ID NO: 15 are useful for transducing motor neurons. In some embodiments, the variant AAV contains a variant capsid protein with an amino acid insertion within loop VIII, the amino acid insertion containing SEQ ID NO: 15. Figures 14E-F show the alignment of residues within loop VIII of the AAV. In some embodiments, loop VIII contains the amino acid positions shown in Table 1.

[0019] [Table 1]

[0020] In some embodiments, loop VIII contains the amino acids corresponding to loop VIII in Figures 14E-F.

[0021] In some embodiments, the amino acid insertion containing SEQ ID NO: 15 is inserted via amino acid substitution at the positions listed in Table 1. In some embodiments, the amino acid insertion containing SEQ ID NO: 15 is inserted via amino acid substitution within loop VIII in Figures 1E-F. In some embodiments, the substitution insertion is further combined with the deletion of one or more amino acids within loop VIII. In some embodiments, the amino acid insertion containing SEQ ID NO: 15 is inserted between two amino acids at the positions in Table 1. In some embodiments, the amino acid insertion containing SEQ ID NO: 15 is inserted between two amino acids within loop VIII in Figures 1E-F. In some embodiments, the insertion is between two discontinuous amino acids (e.g., 573 and 578), and the positions in between (e.g., 574-577) are deleted. In some embodiments, the amino acid insertion includes a substitution at position Q588 of SEQ ID NO: 9 or 14.

[0022] In some embodiments, the amino acid insertion containing SEQ ID NO: 15 further includes a linker. In some embodiments, the amino acid insertion is formula L1-X-L2 (Formula 1) (In the formula, L1 contains the first amino acid linker sequence, X includes sequence number 15, L2 is the second linker sequence. It also includes.

[0023] In some embodiments, L1 and L2 are the same sequence. In some embodiments, the amino acid linker is a glycine-serine linker, e.g., (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1. In some embodiments, the amino acid linker includes a glycine-alanine linker, an alanine-serine linker, or other flexible linkers. In some embodiments, the linker sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or more amino acids.

[0024] In some embodiments, the variant capsid protein is the variant AAV serotype 9 (AAV9) capsid protein. In some embodiments, the AAV9 capsid protein contains the N272A mutation.

[0025] In some embodiments, a variant capsid protein without amino acid insertions has 85% sequence identity with SEQ ID NO: 16. In some embodiments, a variant capsid protein without amino acid insertions has 90% sequence identity with SEQ ID NO: 9. In some embodiments, a variant capsid protein without amino acid insertions has 95% sequence identity with SEQ ID NO: 9. In some embodiments, a variant capsid protein without amino acid insertions has 96% sequence identity with SEQ ID NO: 9. In some embodiments, a variant capsid protein without amino acid insertions has 97% sequence identity with SEQ ID NO: 9. In some embodiments, a variant capsid protein without amino acid insertions has 98% sequence identity with SEQ ID NO: 9. In some embodiments, a variant capsid protein without amino acid insertions has 99% sequence identity with SEQ ID NO: 9. In some embodiments, a variant capsid protein without amino acid insertions has SEQ ID NO: 9.

[0026] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 14. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 14. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 14. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 14. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 14. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 14. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 14. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 14.

[0027] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 1. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 1. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 1. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 1. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 1. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 1. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 1. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 1.

[0028] In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity with SEQ ID NO: 2. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity with SEQ ID NO: 2. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity with SEQ ID NO: 2. In some embodiments, the variant capsid protein without amino acid insertions has 96% sequence identity with SEQ ID NO: 2. In some embodiments, the variant capsid protein without amino acid insertions has 97% sequence identity with SEQ ID NO: 2. In some embodiments, the variant capsid protein without amino acid insertions has 98% sequence identity with SEQ ID NO: 2. In some embodiments, the variant capsid protein without amino acid insertions has 99% sequence identity with SEQ ID NO: 2. In some embodiments, the variant capsid protein without amino acid insertions has SEQ ID NO: 2.

[0029] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 3. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 3. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 3. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 3. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 3. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 3. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 3. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 3.

[0030] In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity with SEQ ID NO: 4. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity with SEQ ID NO: 4. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity with SEQ ID NO: 4. In some embodiments, the variant capsid protein without amino acid insertions has 96% sequence identity with SEQ ID NO: 4. In some embodiments, the variant capsid protein without amino acid insertions has 97% sequence identity with SEQ ID NO: 4. In some embodiments, the variant capsid protein without amino acid insertions has 98% sequence identity with SEQ ID NO: 4. In some embodiments, the variant capsid protein without amino acid insertions has 99% sequence identity with SEQ ID NO: 4. In some embodiments, the variant capsid protein without amino acid insertions has SEQ ID NO: 4.

[0031] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 5. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 5. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 5. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 5. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 5. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 5. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 5. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 5.

[0032] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 6. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 6. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 6. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 6. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 6. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 6. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 6. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 6.

[0033] In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity with SEQ ID NO: 7. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity with SEQ ID NO: 7. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity with SEQ ID NO: 7. In some embodiments, the variant capsid protein without amino acid insertions has 96% sequence identity with SEQ ID NO: 7. In some embodiments, the variant capsid protein without amino acid insertions has 97% sequence identity with SEQ ID NO: 7. In some embodiments, the variant capsid protein without amino acid insertions has 98% sequence identity with SEQ ID NO: 7. In some embodiments, the variant capsid protein without amino acid insertions has 99% sequence identity with SEQ ID NO: 7. In some embodiments, the variant capsid protein without amino acid insertions has SEQ ID NO: 7.

[0034] In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity with SEQ ID NO: 8. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity with SEQ ID NO: 8. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity with SEQ ID NO: 8. In some embodiments, the variant capsid protein without amino acid insertions has 96% sequence identity with SEQ ID NO: 8. In some embodiments, the variant capsid protein without amino acid insertions has 97% sequence identity with SEQ ID NO: 8. In some embodiments, the variant capsid protein without amino acid insertions has 98% sequence identity with SEQ ID NO: 8. In some embodiments, the variant capsid protein without amino acid insertions has 99% sequence identity with SEQ ID NO: 8. In some embodiments, the variant capsid protein without amino acid insertions includes SEQ ID NO: 8.

[0035] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 10. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 10. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 10. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 10. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 10. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 10. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 10. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 10.

[0036] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 11. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 11. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 11. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 11. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 11. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 11. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 11. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 11.

[0037] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 12. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 12. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 12. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 12. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 12. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 12. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 12. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 12.

[0038] In one embodiment, the variant capsid protein without amino acid insertion has 85% sequence identity with SEQ ID NO: 13. In another embodiment, the variant capsid protein without amino acid insertion has 90% sequence identity with SEQ ID NO: 13. In another embodiment, the variant capsid protein without amino acid insertion has 95% sequence identity with SEQ ID NO: 13. In another embodiment, the variant capsid protein without amino acid insertion has 96% sequence identity with SEQ ID NO: 13. In another embodiment, the variant capsid protein without amino acid insertion has 97% sequence identity with SEQ ID NO: 13. In another embodiment, the variant capsid protein without amino acid insertion has 98% sequence identity with SEQ ID NO: 13. In another embodiment, the variant capsid protein without amino acid insertion has 99% sequence identity with SEQ ID NO: 13. In another embodiment, the variant capsid protein without amino acid insertion includes SEQ ID NO: 13.

[0039] In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity to SEQ ID NO: 16. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 16. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity to SEQ ID NO: 16. In some embodiments, the variant capsid protein without amino acid insertions has 96% sequence identity to SEQ ID NO: 16. In some embodiments, the variant capsid protein without amino acid insertions has 97% sequence identity to SEQ ID NO: 16. In some embodiments, the variant capsid protein without amino acid insertions has 98% sequence identity to SEQ ID NO: 16. In some embodiments, the variant capsid protein without amino acid insertions has 99% sequence identity to SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 16.

[0040] In some embodiments, the variant capsid protein includes SEQ ID NO: 15, and the remaining capsid sequence has at least 85% sequence identity with SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 15, and the remaining capsid sequence has at least 90% sequence identity with SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 15, and the remaining capsid sequence has at least 95% sequence identity with SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 15, and the remaining capsid sequence has at least 96% sequence identity with SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 15, and the remaining capsid sequence has at least 97% sequence identity with SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 15, and the remaining capsid sequence has at least 98% sequence identity with SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 15, and the remaining capsid sequence has at least 99% sequence identity with SEQ ID NO: 16. In some embodiments, the variant capsid protein includes SEQ ID NO: 16.

[0041] Percent sequence identity, or simply sequence identity, generally refers to and encompasses the number of identical matched positions shared between two polynucleotide or polypeptide sequences across a comparison window, taking into account any additions or deletions (i.e., gaps) that may be introduced for optimal alignment of the two sequences. A matched position is any position where identical nucleotides or amino acids are presented in both the target and reference sequences. Gaps are not counted because they are neither nucleotides nor amino acids, and therefore gaps presented in the target sequence are not counted. Similarly, gaps present in the reference sequence are not counted because nucleotides or amino acids in the target sequence are counted, rather than those from the reference sequence.

[0042] In some embodiments, the percentage of sequence identity is calculated by determining the number of positions where identical amino acid residues or nucleic acid bases occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be achieved using software readily available in both online and downloadable versions. Suitable software programs are available from various sources for both protein and nucleotide sequence alignment. One suitable program for determining percentage sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). bl2seq compares two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described in Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules described herein. Gapped BLAST can be used to obtain gapped alignments for comparison purposes, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.When using BLAST and Gapped BLAST programs, you can use the default parameters for each program (e.g., XBLAST and NBLAST). See worldwideweb.ncbi.nlm.nih.gov. Other suitable programs include, for example, Needle, Stretcher, ALIGN, Water, or Matcher, some of the bioinformatics programs in the EMBOSS suite, and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0043] Adeno-associated virus, or AAV, or AAV vector, generally refers to and encompasses an assembled viral capsid containing encapsulated nucleic acid molecules. An AAV capsid refers to an assembled viral capsid containing a capsid protein. In some embodiments, the AAV capsid is a functional AAV capsid, for example, capable of being fully folded and / or assembled, capable of infecting target cells (e.g., motor neurons), and / or remaining stable (e.g., remaining folded / assembled and / or capable of infecting target cells). Wild-type AAV capsid protein sequences are encompassed by SEQ ID NOs: 1–13. In some embodiments, the AAV capsid protein contains at least 70% (e.g., 85%, 90%, 95%, or 100%) sequence identity to any one of SEQ ID NOs: 1–13. Figures 14A–G show alignments of AAV capsid protein sequences, with notes on the structural elements of selection.

[0044] Transduction of motor neurons Motor neurons generally refer to and encompass a subset of neurons whose cell bodies are located in the motor cortex, brainstem, or spinal cord, and whose axons extend into or outside the spinal cord, directly or indirectly controlling muscles and glands. In some embodiments, motor neurons are choline acetyltransferase-positive and / or SMI-32-positive. In some embodiments, motor neurons are also NeuN-positive. In some embodiments, motor neurons are upper motor neurons. In some embodiments, motor neurons are lower motor neurons. In some embodiments, motor neurons include both upper and lower motor neurons. In some embodiments, motor neurons are individual. In some embodiments, motor neurons are located within the spine. In some embodiments, motor neurons are located within the anterior horn region of the spinal cord. In some embodiments, motor neurons are located within the cervical to sacral region of the spine. In some embodiments, motor neurons are located within the cervical region of the spine, within the thoracic region of the spine, within the lumbar region of the spine, or within the sacral region of the spine.

[0045] In some embodiments, the variant AAV contains a variant capsid protein with an amino acid insertion within loop VIII, the amino acid insertion containing SEQ ID NO: 15, and exhibits higher transduction (e.g., by vector-mediated transgene expression or by measurement by vector genome per diploid genome equivalent) compared to transduction achievable by an equivalent dose of control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, the variant AAV shows reduced transduction in choline acetyltransferase-negative neurons and non-neuronal cells (e.g., glia) compared to control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0046] In some embodiments, variant AAVs exhibit increased transduction in upper motor neurons (e.g., by vector-mediated transgene expression or by measurement by vector genome per diploid genome equivalent) compared to transduction achievable by an equivalent dose of control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, variant AAVs exhibit decreased transduction in non-neuronal cells (e.g., glia) compared to control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0047] In some embodiments, variant AAV transducers choline acetyltransferase-positive motor neurons at a higher rate than choline acetyltransferase-negative neurons and non-neuronal cells (e.g., glia) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, variant AAV transducers upper motor neurons at a higher rate than other types of neurons and non-neuronal cells (e.g., glia) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0048] In some embodiments, variant AAVs exhibit lower hepatocyte transduction (e.g., measured by vector-mediated transgene expression or vector genome per diploid genome equivalent) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, variant AAVs exhibit lower cardiac cell transduction (e.g., measured by vector genome per diploid genome equivalent) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0049] Pharmaceutical composition A variant AAV is provided in a pharmaceutical composition comprising one or more excipients, wherein the variant AAV comprises a variant capsid protein, the variant capsid protein comprises an amino acid insertion within loop VIII, and the amino acid insertion comprises SEQ ID NO: 15. As used herein, excipients comprise and / or refer to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other component other than the variant AAV vector, and are typically included in formulations and / or administration to a patient. A pharmaceutical composition may comprise a single pharmaceutical formulation or multiple formulations. Excipients further comprise and / or refer to agents that may be added to a formulation to provide a desired consistency (e.g., modify bulk properties), improve stability, and / or adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers. In some embodiments, nonionic excipients, as used herein, comprise and / or refer to agents that have no net charge.

[0050] In some embodiments, nonionic excipients do not have a net charge under certain formulation conditions, such as pH. Examples of nonionic excipients include, but are not limited to, sugars (e.g., sucrose), sugar alcohols (e.g., mannitol), and nonionic surfactants (e.g., polysorbate 80).

[0051] method This specification provides a favorable method for delivering nucleic acid molecules (e.g., therapeutic nucleic acids) to motor neurons. The favorable method utilizes a variant AAV comprising a variant capsid protein having an insertion including sequence number 15, in order to efficiently and effectively transduce motor neurons.

[0052] In some embodiments, methods for delivering nucleic acid molecules to motor neurons of an individual are provided herein, the methods comprising the steps of administering a variant adeno-associated virus (AAV) comprising a nucleic acid molecule to the individual, wherein the variant AAV comprises a variant capsid protein having an amino acid insertion in loop VIII, and the amino acid insertion comprises SEQ ID NO: 15.

[0053] In some embodiments, methods for delivering therapeutic nucleic acid molecules to motor neurons of subjects having motor neuron disease are provided herein, the methods comprising the steps of administering a variant adeno-associated virus (AAV) containing a therapeutic nucleic acid molecule to an individual, wherein the variant AAV contains a variant capsid protein having an amino acid insertion in loop VIII, and the amino acid insertion comprises SEQ ID NO: 15.

[0054] In some embodiments, methods are provided herein for treating a disease or disorder of motor neurons in an individual, the method comprising the steps of administering a variant adeno-associated virus (AAV) containing a therapeutic nucleic acid molecule to the individual, wherein the variant AAV contains a variant capsid protein having an amino acid insertion within loop VIII, and the amino acid insertion comprises SEQ ID NO: 15. Motor neuron disorders generally refer to and encompass diseases that interfere with the normal function of motor neurons. In some embodiments, motor neuron disorders are characterized by muscle atrophy or wasting and / or fasciculations.

[0055] In some embodiments, methods for delivering nucleic acids to motor neurons are provided herein, the methods comprising the steps of bringing a motor neuron into contact with a variant adeno-associated virus (AAV) comprising a nucleic acid molecule, wherein the variant AAV comprises a variant capsid protein having an amino acid insertion in loop VIII, and the amino acid insertion comprises SEQ ID NO: 15.

[0056] In some embodiments, the motor neurons are upper motor neurons. In some embodiments, the motor neurons are lower motor neurons. In some embodiments, the motor neurons include both upper and lower motor neurons. In some embodiments, the motor neurons belong to an individual. In some embodiments, the motor neurons are located within the spine. In some embodiments, the motor neurons are located within the anterior horn region of the spinal cord. In some embodiments, the motor neurons are located within the cervical to sacral region of the spine. In some embodiments, the motor neurons are located within the cervical region of the spine, the thoracic region of the spine, the lumbar region of the spine, or the sacral region of the spine.

[0057] In some embodiments, the variant capsid protein is the AAV serotype 9 (AAV9) capsid protein. In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity to SEQ ID NO: 9. In some embodiments, the variant capsid protein without amino acid insertions contains 90% sequence identity with respect to SEQ ID NO: 9.

[0058] In some embodiments, the AAV9 capsid protein contains the N272A mutation. In some embodiments, the variant capsid protein without amino acid insertions has 85% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 90% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions has 95% sequence identity to SEQ ID NO: 14. In some embodiments, the variant capsid protein without amino acid insertions contains 90% sequence identity with respect to SEQ ID NO: 14.

[0059] In some embodiments, the amino acid insertion further includes a linker. In some embodiments, the amino acid insertion is formula L1-X-L2 (In the formula, L1 contains the first amino acid linker sequence, X includes sequence number 15, L2 is the second linker sequence. It also includes.

[0060] In some embodiments, L1 and L2 are different sequences. In some embodiments, L1 and L2 are the same sequence.

[0061] In some embodiments, the variant AAV contains a variant capsid protein with an amino acid insertion within loop VIII, the amino acid insertion containing SEQ ID NO: 15, and exhibits higher transduction of choline acetyltransferase-positive motor neurons compared to transduction achievable by an equivalent dose of control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, the variant AAV exhibits reduced transduction of choline acetyltransferase-negative neurons and non-neuronal cells (e.g., glia) compared to control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0062] In some embodiments, variant AAVs exhibit increased transduction in upper motor neurons compared to transduction achievable by an equivalent dose of control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, variant AAVs exhibit decreased transduction in non-neuronal cells (e.g., glia) compared to control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0063] In some embodiments, variant AAV transducers choline acetyltransferase-positive motor neurons at a higher rate than choline acetyltransferase-negative neurons and non-neuronal cells (e.g., glia) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, variant AAV transducers upper motor neurons at a higher rate than other types of neurons and non-neuronal cells (e.g., glia) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0064] In some embodiments, variant AAVs exhibit lower hepatocyte transduction (e.g., measured by vector genome per diploid genome equivalent) compared to control AAVs (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9). In some embodiments, variant AAVs exhibit lower cardiac cell transduction (e.g., measured by vector genome per diploid genome equivalent) compared to control AAVs (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

[0065] In some embodiments, variant AAV is administered by injection into the individual. In some embodiments, the injection is intravenous.

[0066] As used herein, individual is synonymous with patient and / or subject, and includes and / or refers to a human being who has been diagnosed with a disease or illness disclosed herein and may require treatment. However, it is not limited to humans, and examples include chimpanzees, marmosets, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and so on. Individual is typically a human being who has been diagnosed with a disease or illness disclosed herein and may require treatment.

[0067] As used herein, treating or treating includes and / or means improving a disease or disorder or its symptoms (e.g., slowing, stopping or reducing the onset of at least one of the disease or its clinical symptoms). In some embodiments, treating or treating also includes and / or means mitigating or improving at least one physical and / or biological parameter, including one that may not be identifiable by the patient. In some embodiments, treating or treating includes and / or means regulating a disease, disorder, or biological process either physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical and / or biological parameters), or both. In some embodiments, treating or treating includes and / or means preventing or delaying the onset, onset, or progression of a disease or disorder. In certain embodiments, treating or treating includes and / or means preventing, delaying, or inhibiting (i) a healthy physiological state or (ii) a deterioration of a baseline physiological state (e.g., progression of a disease or disorder).

[0068] As used herein, a sample includes and / or refers to any fluid or liquid sample that is analyzed to detect and / or quantify the analyte. In some embodiments, a sample is a biological sample. Examples of samples, but not limited to, include body fluids, extracts, solutions containing proteins and / or DNA, cell extracts, cell lysates, or tissue lysates. Non-limited examples of body fluids include urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen, sweat, pleural fluid, liquefied feces, and lacrimal gland secretions.

[0069] As used herein, “a” or “an” means, when used in conjunction with the term “comprising” in the claims and / or specification, “one” and / or refers to “one,” and is also consistent with the meanings of “one or more,” “at least one,” and “one or more than one.” Similarly, the term “another” may mean at least two or more.

[0070] As used herein, the terms “comprising” (all forms of comprising, such as comprise and comprises), “having” (and all forms of having, such as have and has), “including” (and all forms including include and includess), or “containing” (and all forms including contain and containss) are inclusive or non-exclusive and do not exclude additional unlisted elements or steps of a process. As used herein, in any example or embodiment, “comprising” may be replaced with “consisting essentially of” and / or “consisting of.” As used herein, in any example or embodiment, “comprises” may be replaced with “consists essentially of” and / or “consists of.”

[0071] As used herein, the term “about” in the context of a given value or range includes and / or refers to a value or range that is within 20%, within 10%, and / or within 5% of a given value or range.

[0072] Where used herein, the term “and / or” should be interpreted as a specific disclosure of each of two identified features or components, with or without the other. For example, “A and / or B” should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were described separately herein. [Examples]

[0073] Example 1 - Motor neuron targeting AAV-CAP2 is an adeno-associated virus (AAV) containing nucleic acid molecules into an organism, and this variant AAV contains a variant capsid protein, which contains an amino acid insertion within loop VIII, the amino acid insertion containing SEQ ID NO: 15 (e.g., SEQ ID NO: 16), and is a novel AAV capsid identified by the TRANnscription-dependent Directed Evolution (TRADE) platform, exhibiting enhanced motor neuron tropism after vector administration in both rodents and non-human primates (NHPs).

[0074] Cynomolgus monkeys and C57BL / 6 mice were treated with a single intravenous injection of the AAV-CAP2-CAG-GFP vector (an AAV vector containing a nucleic acid molecule encoding GFP and a variant capsid protein with an amino acid insertion within loop VIII). Data were collected from the animals 3 weeks after injection, and tissues were collected for downstream histological and in vivo distribution analysis. The efficiency and specificity of motor neuron transduction by the CAP2 variant were evaluated by characterizing the phenotype of GFP-positive transdextrins with triple immunolabeling using selective pan-neuronal markers (NeuN, neuronal nuclei) and motor neuron markers (SMI-32 and choline acetyltransferase, ChAT). Based on previous histological descriptions, Betz cells in the NHP primary motor cortex were identified by the following set of criteria: their cell body size (>600 μm), localization (sublayer 5b), proximal dendritic morphology, and SMI-32 immunoreactivity.

[0075] AAV-CAP2 showed a significant bias in transducing SMI-32-immunopositive upper motor neurons, with minimal glial transduction in both species. In the anterior horn of NHP, ChAT immunostaining revealed that numerous motor neurons homogeneously expressed GFP along the entire anterior horn from the cervical to the lumbar segment. GFP signaling was also present in sciatic nerve axons, indicating transport of GFP protein along the peripheral axonal pathway. GFP-positive motor neurons were counted in the cervical and lumbar spinal dilatae, innervating the upper and lower limbs, respectively. Quantification of motor neurons revealed that >80% of lower motor neurons expressed GFP in the cervical and lumbar spinal segments of vector-injected NHP. Notably, no clear GFP signaling was detected in ChAT-negative cells, supporting the motor neuron tropism of the CAP2 variant. Spinal motor neuron transduction was also evident in mice, but to a lesser extent compared to NHP, suggesting potential interspecies differences. The data demonstrate that AAV-CAP2 is a potent vector for use in the treatment of a wide range of motor neuron diseases.

[0076] the purpose The initial challenge was to develop a novel AAV capsid capable of transducing neurons in the brain and spinal cord via intravenous (iv) delivery. AAV9 was selected as the backbone. The AAV9 backbone was modified with the N272A mutation, which had previously been shown to confer a liver-nontargeting phenotype (data not shown). The pAAV9-N272A-hSYN1-TRADE plasmid was constructed to contain two expression cassettes oriented antisense toward each other and flanked by AAV2 reverse terminal repeat sequences (ITRs). At one end of the genome, the AAV2 p40 promoter and AAV2 viral genomic intron are followed by the AAV9-N272A cap gene and the AAV2 polyadenylation signal. For antisense against the p40-driven cap ORF on the opposite side of the genome, an overlapping transgene cassette was used, consisting of a human synapsin I (hSYN1) promoter-enhancer sequence, a mouse cleaved microvirus (MVM) intron, a non-coding antisense cap sequence, and an SV40 polyadenylation signal located in the AAV2 intron (Figure 1). The hSYN1 promoter-enhancer element was selected to drive the expression of antisense mRNA in neurons in vivo, and its expression can be used to identify capsid variants exhibiting enhanced neurotropy. The initial TRADE peptide display plasmid library (pAAV9-N272A-hSYN1-TRADE-Lib0) was constructed by PCR amplification of an AAV9 cap sequence with a loop VIII[NNK]8 (GGGS[NNK]8GGGGS) substitution at position Q588 of AAV9N272A VP1. The plasmid library was used to construct the corresponding viral library using a standard triple transfection production method. Figure 1 shows an illustration of the TRADE structure.

[0077] In Vivo Selection The initial in vivo selection was performed in 8-week-old C57BL / 6J male mice. Two mice were administered the AAV9-N272A-hSYN1-TRADE-Lib0 vector, and brain tissue was collected 12 days after injection. For this first round of selection, relatively neuronal-enriched brain regions were used; therefore, RNA was extracted from the crudely dissected frontal cortex. Total RNA was isolated independently from each animal, mixed in a 1:1 ratio, and the recovered sequences were identified by RT-PCR. Regions containing peptide insertions were assembled in the TRADE vector backbone to generate a plasmid library (i.e., pAAV9-N272A-hSYN1-TRADE-Lib1) representative of the capsid sequences that underwent one round of selection. This plasmid library was used to construct the corresponding AAV library, and this process was repeated for a total of three selections in mice. Rounds 2 and 3 were performed in a similar manner to the first round, with some modifications. In the latter round, 1 × 10⁶ dilutions were used per mouse. 9 ~1 × 10 12 A dose within the range of vg was administered (n=2 animals per dose), but 1 × 10 11 Only samples from animals administered the vg dose were carried over. In parallel, a single selection was performed using male rhesus macaques. The AAV9-N272A-hSYN1-TRADE-Lib0 vector was administered intravenously, and the animals were euthanized 14 days after injection. Tissues were collected and frozen on dry ice. RT-PCR amplicons of antisense cap mRNA containing peptide fragments were recovered from RNA extracted from the brain, subcloned into a plasmid backbone, and formed a plasmid library. Subsequently, DH10B cells were transformed with the plasmid library, individual clones were miniprepped, and sent for Sanger sequencing. After three rounds of selection in C57BL / 6J mice, the top five most common variants, as well as 21 variants recovered by Illumina sequencing after one round of selection from various regions of the rhesus macaque brain, were selected for AAV DNA / RNA Barcode-Seq analysis.

[0078] AAV DNA / RNA Barcode-Seq Analysis Using a standard triple transfection method, viral barcode clones were individually packaged into novel, TRADE-identified capsids. Furthermore, barcoded vectors were constructed in key reference capsids containing AAV9, AAV9-N272A (parent), and AAV-PHP.B. Crude lysate titers of the DNase-resistant vector genomes were determined by quantitative dot-blotting assays using probes against the hSYN1 promoter. Each barcoded viral clone was then mixed in approximately equimolar ratios based on dot-blotting titer, and the pooled crude lysates were purified as a library to produce scAAV-hSYN1-GFP-BCLib.

[0079] scAAV-hSYN1-GFP-BCLib was administered to one male rhesus monkey in 2x10 13 The mice were administered at a dose of vg / kg and given 1.7 x 10¹⁶ doses to C57BL / 6J mice (n=3). 13 The drug was administered at a dose of vg / kg, and tissue samples were collected two weeks after injection. AAV RNA barcode-seq was performed on tissues sectioned from various brain regions. From this analysis, AAV-CAP2 was selected for further characterization (data not shown).

[0080] AAV-CAP2 single capsid validation in NHP To evaluate the ability of AAV-CAP2 to transduce neurons, enhanced green fluorescent protein (GFP) under the regulation of a ubiquitous CAG promoter (enabling transcellular characterization throughout the body) was packaged within AAV-CAP2. The adopted GFP reporter possesses an N-terminal nuclear localization signal (nls) that allows for some nuclear enrichment while preserving cytoplasmic signaling. Two cynomolgus monkeys were transduced to 5 × 10⁶ 13Animals were treated with a single intravenous injection of the AAV-CAP2-CAG-GFP vector at a dose of vg / kg. Three weeks after injection, the animals were euthanized, and tissue was collected for downstream in vivo distribution analysis and histological analysis. Immunostaining with anti-GFP antibody revealed robust and widespread GFP expression across multiple brain regions and neuronal subtypes in the brains of adult NHPs, with the strongest expression observed in the motor cortex and thalamic regions (Figure 2A-B). Double immunofluorescence microscopy analysis was performed, coupling GFP expression with the panneuronal marker NeuN (neuronal nucleus) to determine the neuronal transduction efficiency and specificity of transdextrins (Figure 2B). This analysis revealed that transdextrins accounted for 93% of neurons in the motor cortex, 90% of neurons in the striatum, and 97% of neurons in the thalamus and lateral geniculate nucleus (Figure 2C). Glial transduction was minimal, as measured by co-staining with the astrocytocyte marker GFAP and the oligodendrocyte marker Olig2 (data not shown).

[0081] Upper motor neuron phenotype induction by AAV-CAP2 Interestingly, in the prefrontal cortex, most of the transfected cells were localized throughout layer Vb of the primary motor cortex and exhibited upper motor neuron morphology (Figure 3A). Upper motor neurons (UMNs) refer to corticospinal neurons that synapse with Betz giant pyramidal cells in NHP and humans, or lower motor neurons (LMNs) in the ventral horn of the spinal cord in mice (Menon and Vucic 2021; Braak and Braak 1976; 2003). Based on previous histological descriptions, Betz cells in the NHP primary motor cortex can be identified by a set of the following criteria. Their cell body size (>600 μm), localization (sub-layer Vb), proximal dendritic morphology, and SMI-32 immunoreactivity (Braak and Braak 1976; Jacobs et al. 2018; Meyer 1987; Rivara et al. 2003; Szocsics et al. 2021). To evaluate the efficiency and specificity of upper motor neuron / Betz cell transfection, morphometric analysis of GFP-positive cells in layer Vb of the primary motor cortex was performed (Figure 3B). The distribution of the somatic cell sizes of the transfected cells showed a bimodal pattern, with one peak at 200 - 300 μm 2 and another peak at 800 - 900 μm 2 (Figure 3B). When making a categorical distinction of somatic cell sizes between Betz cells and pyramidal cells at 600 μm 2 most of the transfected cells in layer Vb of the motor cortex were 600 - 1300 μm 2The somatic cell sizes were within the specified range (Figure 3B). Next, the phenotypes of these large GFP-positive transducers were characterized in the motor cortex by dual immunofluorescence microscopy for GFP and the selective upper motor neuron marker SMI-32 (Figure 3C). Surprisingly, AAV-CAP2 transduced 48% of all large SMI-32-positive Betz cells in the NHP motor cortex, and 71% of all transducers in layer Vb were SMI-32-positive Betz cells. The vector copy number in the motor cortex and putamen was determined for both animals using ddPCR (Figure 4A). GFP quantification data obtained by ddPCR were validated by quantifying genomic references at the ribonuclease P protein subunit p30 (RPP30) locus to determine the vector genome copy number per diploid genome. The mean vector genome copy number per diploid genome in the cortex and putamen was 0.4 and 0.2, respectively (Figure 4A). While data on motor neuron transduction or in vivo distribution are not available in the literature, a recent publication (Stanton et al. 2023) reported 3 × 10⁻⁶ 13 We reported a vector copy count of 0.05 in the motor cortex after IV administration of AAV9 at a dose of vg / kg (Figure 4B). 3×10 13 When the vg / kg dose was used in the study, the estimated copy number corresponds to 0.009 vg / diploid genome, which is about four times lower than what would be observed assuming a linear dose response. Considering that AAV9 transduces more glial cells than neurons in the brains of NHP patients, the estimated copy number in neurons in NHP brains in Stanton et al.'s study should be less than 0.009 vg / diploid genome, highlighting the superiority of AAV-CAP2 over AAV9 in neuronal transduction in NHP patients.

[0082] Lower motor neuron transduction using AAV-CAP2 Considering the increased efficacy of AAV-CAP2 in transduction of upper motor neurons, we investigated the ability of AAV-CAP2 to enhance transduction in the spinal cord. A single intravenous injection of AAV-CAP2-CAG-GFP induced neuronal transduction along the entire length of the spinal cord. Robust GFP signaling was detected in cells with motor neuron morphology and their axons at the cervical (Figure 5B, F, and E), thoracic (Figure 5C and G), and lumbar (Figure 5D, H, and I) levels.

[0083] To identify these GFP-positive cells as lower motor neurons, immunostaining was performed with ChAT (choline acetyltransferase), an enzyme that catalyzes acetylcholine synthesis and acts as a selective marker for cholinergic motor neurons in the anterior horn (Stifani 2014). ChAT immunostaining demonstrated that the majority of motor neurons express GFP in both the cervical (81%) and lumbar (83%) segments innervating the upper and lower limbs, respectively (Figure 6A-C) (Stifani 2014).

[0084] GFP transgene-specific quantitative PCR performed in the cervical, thoracic, and lumbar spinal segments of two injected macaques showed equal or higher numbers of vg per diploid genome in the spinal cord compared to those reported in the literature (Gray et al. 2011; Hinderer et al. 2018) (Figure 7).

[0085] Transduction of dorsal root ganglia by AAV-CAP2 High transduction of DRGs after AAV vector administration has been associated with neuroinflammation that can lead to severe neurotoxicity. DRG transduction was evaluated 21 days after intravenous administration of AAV-CAP2-CAG-GFP (Figure 8). Immunostaining with antibody against GFP (green) and the neuronal marker NeuN (red) showed an average transduction of 7%–15% in DRGs throughout the spine (Figures 8A and B). The vector genome copy number per diploid genome in DRGs was determined to be 0.05–0.03 (Figure 8C).

[0086] The spinal cords and DRGs of two NHPs were evaluated for abnormal pathologies, including mononuclear cell infiltration, neuronal cell body degeneration in the DRGs, and secondary axonal damage in the spinal cords. Immune responses in multiple DRG sections from the cervical, thoracic, and lumbar regions of the vertebrae of both animals were analyzed by experienced histopathologists on hematoxylin and eosin (H&E) stained histological sections. Severity scores for each individual's DRG segment were established using a scale developed by Hordeaux (Hordeaux et al. 2020). Only mild lymphocyte infiltration was reported in the DRGs, and secondary axonal damage was not detected in the spinal cords of either animal (data not shown).

[0087] Biological distribution in peripheral organs From the same animals, liver and heart portions were used for in vivo distribution analysis and quantitative PCR (qPCR and ddPCR) quantification of the vector genome (Figure 9A). Comparison of liver vector genome copy number data with literature-reported data (Figure 9B) showed that AAV-CAP2 demonstrated lower vector genome abundance in the liver compared to AAV9, with the exception of a study by Gray et al. (Gray et al. 2011) which reported a surprisingly low liver vector genome copy number. (Gray et al. 2011; Hinderer et al. 2018; Horiuchi et al. 2022; Meseck et al. 2022)

[0088] Verification of AAV-CAP2 single capsid in mice Our findings demonstrate that broad and potent upper and lower motor neuron transduction is achievable by a single intravenous injection of AAV-CAP2 in NHP. To determine whether systemic delivery of AAV-CAP2 in mice also results in enhanced neuronal transduction similar to that observed in NHP, we conducted a 5x10⁻¹⁰ study. 13 AAV-CAP2-CAG-GFP at a dose of vg / kg was injected via the tail vein into 8-week-old C57BL / 6 male mice. Three weeks after injection, tissue samples were collected and evaluated for neuronal transduction and vector biodistribution. The results showed that the AAV-CAP2 vector transduced a high fraction of neurons (measured by co-staining with NeuN) in all tested brain regions across the mouse central nervous system (CNS). The rate of neuronal transduction varied between regions; 8% of neurons in the motor cortex colocalized with the GFP signal, and 25% in the thalamus, but this percentage decreased to 3% in the striatum (putamen) (Figure 10A). The high neuronal specificity attribute of AAV-CAP2 was also retained in mice, showing 80–99% neuronal specificity across different regions (Figure 10B).

[0089] We investigated whether the motor neuron tropism of the AAV-CAP2 vector, as observed in NHP, is maintained in mice. Equivalent upper and lower motor neuron transduction analyses were performed in mice. First, AAV-CAP2-CAG-GFP transdescent cells with neuronal morphology along the "motor strip" in layer Vb of the mouse primary motor cortex were observed (Figure 11A). This profile was similar to the findings in NHP. Further upper motor neuron transduction efficiency analysis confirmed that the target specificity of the AAV-CAP2 vector is biased towards SMI-32 immunopositive upper motor neurons in mice as well (Figure 11B). AAV-CAP2 targeted 25% of SMI-32 positive upper motor neurons, while 45% of all transdescent cells in the mouse primary motor cortex were upper motor neurons (Figures 11B-D).

[0090] Next, we investigated the target specificity of AAV-CAP2 in the mouse spinal cord (Figure 12). Triple staining with GFP, ChAT, and NeuN revealed the colocalization of GFP and NeuN in the anterior horn of ChAT-immunolabeled motor neurons (MNs) (Figure 12A). Scoring of GFP-positive motor neurons in the cervical and lumbar segments showed that 30% of cervical spinal motor neurons and 41% of lumbar spinal motor neurons were transduced in mice. Enhancement of spinal motor neuron transmission was observed, although lower in mice compared to NHP (approximately 80%). This observation may indicate some interspecies differences in transduction.

[0091] Comparison of AAV-CAP2 transduction in mice and NHP The target specificity of the AAV-CAG-CAP2-GFP vector was analyzed in the brains of mice and cynomolgus monkeys. Similar levels of transduced neurons were observed in the brains of mice and NHPs (Figure 13A). Furthermore, the efficiency of upper motor neuron transduction was similar between the two species, further supporting the motor neuron tropism of this capsid (Figure 13B).

[0092] Consideration Upper and lower motor neurons are distinct neuronal subtypes that exhibit primary neurodegeneration in motor neuron diseases. Therefore, efficient transduction of upper and lower motor neurons is crucial for any successful gene therapy approach. Systemic administration of viral vectors may serve as an optimal route of administration; however, inefficient crossing of the blood-brain barrier, poor target cell specificity, and high hepatic uptake remain challenges for commonly used AAV serotypes. Targeting upper motor neurons is particularly difficult given their relatively low abundance in the brain and their specific location within layer Vb of the cortex. Retrograde labeling studies in mice have shown that it is indeed possible to retrogradely transduce motor neurons using AAV to induce selective gene expression; however, these approaches have considerable limitations as they target only motor neurons innervating specific muscle groups (Genc et al. 2022; Jara et al. 2014). Furthermore, there are currently no approaches for selective Betz cell transduction in NHP and human motor cortex. In this respect, AAV-CAP2 shows a significant bias towards transducing upper motor neurons with minimal glial transduction in both mice and NHP.

[0093] Efficient systemic spinal cord transduction to lower motor neurons is also central to the success of gene therapy and genetic manipulation of motor neuron diseases. Previous research (Hinderer et al. 2018) showed that 30–90% of motor neuron transduction at each level of the spinal cord in rhesus monkeys occurs at 2 × 10⁶ 14It has been reported that the AAV9 variant AAVhu68 can be achieved after systemic administration at doses of vg / kg, which is four times higher than the dose used in this study. However, unexpected toxicities, including acute systemic inflammation, coagulation disorders, and hepatotoxicity, occurred after such high doses (Hinderer et al. 2018). Intrathecal delivery has been shown to be less affected by pre-existing antibodies, although pre-existing antibodies are lower in the cerebrospinal fluid. Therefore, the intrathecal approach is employed for transduction of NHP in the brain and spinal cord, but transduction efficiency still needs to be improved to reach therapeutic levels (Gray et al. 2013). Intrathecal lumbar injection of another AAV9 variant, AAV-F, into cynomolgus monkeys resulted in lower motor neuron transduction gradients ranging from 70% to 40% from the lumbar region (higher) to the cervical region (lower), respectively (Beharry et al. 2022). In mice, IV delivery of AAV9 in adult animals achieved a motor neuron transduction rate that was below average at approximately 19% (Duque et al. 2009). 13 The observation that intravenous delivery of the vg / kg AAV-CAP2 vector resulted in transduction of approximately 35% of mouse spinal motor neurons and approximately 80% of NHP spinal motor neurons with low toxicity makes AAV-CAP2 an attractive capsid for use in gene therapy to treat motor neuron diseases. The molecular mechanisms of motor neuron tropism of the AAV-CAP2 vector are currently unknown, but they may arise from a combination of differences in capsid amino acids and interactions with specific receptors or co-receptors that differ from those of other AAV capsids, which requires further investigation.

[0094] In summary, the above study identified AAV-CAP2, a novel AAV capsid that transduces both upper and lower motor neurons with high efficiency and specificity in both mice and NHP, using TRANscription-dependent Directed Evolution (TRADE). The data suggest that AAV-CAP2 offers a best-in-class capsid for treating a wide range of motor neuron disorders.

[0095] method Non-human primates: Non-human primate studies were conducted at Envoi Biomedical (Florida, USA) using standard operating protocols and procedures approved by the IACUC. At Envoi Biomedical, two healthy, untreated cynomolgus macaques (one male and one female), approximately two years old and housed under standard conditions, were selected for the AAV-CAP2 study. Both animals were confirmed to have serum AAV2 and AAV9 neutralizing antibody titers less than 1:5. The macaques were given 5 × 10⁶ doses of AAV-CAP2-CAG-GFP. 13 An intravenous bolus injection was administered at a dose of vg / kg. The animals were euthanized, perfused with PBS, and then the CNS and other organ tissues were collected. The brain was removed, hemispherectomy was performed on one hemisphere, fixed overnight in 4% paraformaldehyde, and the other hemisphere was dissected to isolate the brain region. The CNS region, as well as peripheral organs, were rapidly frozen and stored at -80°C for molecular processing.

[0096] NHP Neurohistological Embedding, Sectioning, and Staining: At NeuroScience Associates (Knoxville, Tennessee), cerebral hemispheres of two NHPs, as well as all segments of the spinal cord and DRG, were processed for embedding and sectioning. Samples were treated overnight with 20% glycerol and 2% dimethyl sulfoxide in phosphate-buffered saline (PBS) to prevent freeze artifacts. Subsequently, two cerebral hemispheres from the two animals were embedded in the specimen for coronal embedding, 16 coding segments for transverse and coronal embedding, and 8 DRG segments were also embedded. Samples were placed in a gelatin matrix of each block using MultiBrain® / MultiCord® Technology. MultiBrain® / MultiCord® blocks were sectioned using a 40 μm microtome setting.

[0097] DAB-GFP Immunohistochemistry: For immunohistochemistry (IHC), brain sections were stained every 24 sections (960 micron intervals), spinal cord sections every 20 sections (800 micron intervals), and DRG sections every 10 sections (400 micron intervals) using the free flotation method. All incubation solutions after the primary antibody were prepared using Tris-buffered saline (TBS) containing Triton X100 as the vehicle; all rinsing was performed with TBS. After hydrogen peroxide treatment and rinsing, the sections were immunostained with the primary antibody overnight at room temperature, as shown in the table below. The vehicle solution contained Triton X100 for permeabilization. After rinsing, biotinylated secondary antibody (anti-IgG from the host animal that produced the primary antibody) was applied. After further rinsing, Vector Lab's ABC solution catalog number PK-6100 (avidin-biotin-HRP complex; 1:222 dilution) was applied. The sections were rinsed again and then treated with a chromogen, diaminobenzidine tetrahydrochloride (DAB), nickel(II) sulfate, and hydrogen peroxide to prepare a visible reaction product. After further rinsing, the sections were placed on gelatin-coated glass slides and then air-dried. The mounted slides were counterstained with complete Thionine Nissl stain.

[0098] Complete thionine counterstaining: Air-dried, mounted stained slides were treated in the following order: 95% ethanol, 95% ethanol / formaldehyde; 95% ethanol, 70% ethanol, deionized water (dH2O). The slides were then stained with thionine solution prepared in acetate buffer, pH 4.5, rinsed in dH2O, and visually evaluated. After rinsing with dH2O, the slides were dehydrated in alcohol, cleared in xylene, and covered with Permount (Fisher Scientific, Pittsburgh PA) coverslips.

[0099] Mice: Healthy, untreated male C57BL / 6J (JAX, #000664) mice, 8 weeks old, were purchased from Jackson Laboratory. The mice were housed under standard conditions according to the IACUC at Oregon Health and Science University. All mice were administered AAV vector via tail vein injection. Tissue samples were collected from the mice after systemic perfusion with either phosphate-buffered saline (PBS) alone or PBS followed by 4% paraformaldehyde (PFA), depending on downstream experiments.

[0100] Mouse neurohistochemistry, embedding, and sectioning: Brain and spinal cord were sampled with DRG and fixed overnight at 4°C in 4% PFA. The brain was sectioned to a thickness of 40 μm in the coronal position using a Vibratome (Leica VT1000, Wetzlar, Germany) and retained as free-floating sections. Spinal cord and DRG sections were cut to a thickness of 40 μm in the coronal and transverse sections on a cryostat (Microm HM550, Thermo Fisher Scientific, Waltham, MA) and placed on aminosilane-coated slides.

[0101] Immunofluorescence staining: Sections were washed three times with TBS. They were then incubated at room temperature for 1 hour with 0.3% Triton X-100 in TBS containing 5% normal goat serum (NGS), followed by overnight incubation at 4°C with primary antibody diluted in 2% NGS and 0.3% Triton X-100 in TBS. Next, the sections were washed three times with PBS (3 × 10 min) and incubated at room temperature for 1 hour with secondary antibody diluted in PBS. Finally, they were washed with PBS (3 × 10 min) and mounted using DAPI Fluoromount-G (SouthernBiotech). Control sections were stained using the same protocol but without the primary antibody. All processes were performed in a darkroom.

[0102] The primary antibodies used were as follows: chicken anti-GFP (AVES, AB2307313, 1:500), rabbit anti-NeuN (Millipore, ABN78, 1:500), goat anticholine acetyltransferase (ChAT; Millipore, AB144P, 1:100), mouse anti-SMI-32 (Biolegend, 801702, 1:100), and rabbit anti-GFAP (DAKO, Z0334, 1:1000). The secondary antibodies used were as follows: goat anti-chicken immunoglobulin G (IgG; Abcam, Alexa488), goat anti-rabbit IgG (Molecular Proves, Alexa568), goat anti-rabbit IgG (Molecular Proves, Alexa647), goat anti-mouse IgG (Molecular Probes, Alexa555, Eugene, Oregon, USA), and donkey anti-goat IgG (Molecular Probes, Alexa647) (1:300).

[0103] Transduction imaging and quantification: For imaging, an LSM 9MP laser scanning microscope (Carl Zeiss MicroImaging, Thornwood, New York) was used. All quantitative analyses were performed in a blinded manner. Virus directional analysis was performed using the Cell Counter probe in Imaged and Imaris software. Images were acquired from immunofluorescently labeled level-matched sections (4–8 per animal) across the motor cortex, putamen, thalamus, spinal cord, and DRG segments using a 20x lens with equivalent acquisition settings. Cell tropism is reported as a percentage of all GFP-positive cells counted (minimum 50 cells per replica). Statistical analysis was based on the average number per animal. All statistical analyses were performed using Prism software (GraphPad Software Inc., La Jolla, California, USA).

[0104] Quantification of vector genome copy number: Genomic DNA was extracted and purified from different tissues (i.e., brain, spinal cord, DRG, liver, and heart) using the QIAamp DNA tissue kit (Qiagen, Hilden, Germany). The extracted DNA was analyzed for yield and purity using a NanoDrop One UV / Vis spectrophotometer (Thermo Scientific, Wilmington, Massachusetts, USA).

[0105] To measure the viral genome copy number, 1 μg of gDNA was digested with Bam HI without cleaving the viral genome. The digested gDNA samples were diluted to 100 ng, 50 ng, 25 ng, and 12.5 ng. 20 μL of ddPCR reaction mixture was prepared for each dilution using a 2× digital PCR supermix for the probe (without dUTP) and primers / probes targeting the GFP region (VIC) and the RPP30 gene (FAM). The GFP quantification data obtained by ddPCR was confirmed by quantifying the genome reference at the ribonuclease P protein subunit p30 (RPP30) locus to determine the vector genome copy number per diploid genome. The average of all four dilutions was plotted.

[0106] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

Claims

1. A method for delivering nucleic acid molecules to motor neurons of an individual, A step of administering a variant adeno-associated virus (AAV) containing the nucleic acid molecule to the individual, wherein the variant AAV contains a variant capsid protein with an amino acid insertion in loop VIII, and the amino acid insertion contains SEQ ID NO:

15. A method that includes this.

2. A method for delivering therapeutic nucleic acid molecules to motor neurons of a subject with motor neuron disease, A step of administering a variant adeno-associated virus (AAV) containing the therapeutic nucleic acid molecule to the individual, wherein the variant AAV contains a variant capsid protein with an amino acid insertion in loop VIII, and the amino acid insertion includes SEQ ID NO:

15. A method that includes this.

3. The method according to any one of claims 1 or 2, wherein the motor neuron is an upper motor neuron or a lower motor neuron.

4. The method according to any one of claims 1 to 3, wherein the motor neurons are located in the frontal cortex of the brain.

5. The method according to any one of claims 1 to 4, wherein the motor neuron is located within the spinal cord.

6. The method according to any one of claims 1 to 5, wherein the motor neuron is located within the anterior horn region of the spinal cord.

7. The method according to any one of claims 1 to 6, wherein the motor neuron is located within the cervical region to the sacral region of the spine.

8. The method according to any one of claims 1 to 7, wherein the motor neuron is located within the cervical region of the spine, within the thoracic region of the spine, within the lumbar region of the spine, or within the sacral region of the spine.

9. A method for treating motor neuron disease in an individual, A step of administering a variant adeno-associated virus (AAV) containing a therapeutic nucleic acid molecule to the individual, wherein the variant AAV contains a variant capsid protein with an amino acid insertion in loop VIII, and the amino acid insertion contains Sequence ID No.

15. A method that includes this.

10. The method according to claim 9, wherein the variant AAV delivers therapeutic nucleic acid to motor neurons.

11. The method according to claim 10, wherein the motor neuron is an upper motor neuron or a lower motor neuron.

12. The method according to any one of claims 10 or 11, wherein the motor neurons are located within the frontal cortex of the brain.

13. The method according to any one of claims 10 to 12, wherein the lower motor neurons are located within the anterior horn region of the spinal cord.

14. The method according to any one of claims 10 and 12 to 13, wherein the motor neuron is located within the spine.

15. The method according to any one of claims 10 and 12 to 14, wherein the motor neuron is located in the area from the cervical region to the sacral region of the spine.

16. The method according to any one of claims 10 and 12 to 15, wherein the motor neuron is located within the cervical region of the spine, within the thoracic region of the spine, within the lumbar region of the spine, or within the sacral region of the spine.

17. A method for delivering nucleic acids to motor neurons, A step of contacting the motor neuron with a variant adeno-associated virus (AAV) containing nucleic acid molecules, wherein the variant AAV contains a variant capsid protein with an amino acid insertion in loop VIII, and the amino acid insertion includes sequence number 15. A method that includes this.

18. The method according to any one of claims 1 to 17, wherein the variant capsid protein is AAV serotype 9 (AAV9) capsid protein.

19. The method according to claim 18, wherein the AAV9 capsid protein contains the N272A mutation.

20. The method according to any one of claims 1 to 19, wherein the variant capsid protein without the amino acid insertion has 90% sequence identity with respect to SEQ ID NO:

9.

21. The method according to any one of claims 1 to 19, wherein the variant capsid protein without the amino acid insertion has 90% sequence identity with respect to SEQ ID NO:

14.

22. The method according to any one of claims 1 to 21, wherein the amino acid insertion further comprises a linker.

23. The aforementioned amino acid insertion, formula L1-X-L2 (In the formula, L1 contains the first amino acid linker sequence, X contains sequence number 15, (L2 is the second linker sequence.) The method according to any one of claims 1 to 22, further comprising:

24. The method according to claim 23, wherein L1 and L2 are different sequences.

25. The method according to claim 23, wherein L1 and L2 are the same sequence.

26. The method according to any one of claims 1 to 25, wherein the motor neuron is choline acetyltransferase positive.

27. The method according to any one of claims 1 to 26, wherein the motor neuron is SMI-32 positive.

28. The method according to any one of claims 1 to 27, wherein the variant AAV exhibits higher transduction of choline acetyltransferase-positive motor neurons compared to transduction achievable by an equivalent dose of a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

29. The method according to any one of claims 1 to 28, wherein the variant AAV exhibits reduced transduction of choline acetyltransferase-negative neurons and non-neuronal cells (e.g., glia) in comparison to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

30. The method according to any one of claims 1 to 29, wherein the variant AAV exhibits increased transduction of upper motor neurons compared to transduction achievable by an equivalent dose of a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

31. The method according to any one of claims 1 to 30, wherein the variant AAV exhibits reduced transduction of non-neuronal cells (e.g., glia) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

32. The method according to any one of claims 1 to 31, wherein the variant AAV transduces choline acetyltransferase-positive motor neurons at a higher rate than choline acetyltransferase-negative neurons and non-neuronal cells (e.g., glia) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

33. The method according to any one of claims 1 to 32, wherein the variant AAV transduces upper motor neurons at a higher rate than other types of neurons and non-neuronal cells (e.g., glia) compared to a control AAV (e.g., AAV9) containing wild-type AAV9 capsid protein (SEQ ID NO: 9).

34. The method according to any one of claims 1 to 33, wherein the variant capsid comprises SEQ ID NO: 15 and has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 16.

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