A method for screening AAV viral particle libraries in organoids

Abstract

The present invention relates to a method for screening AAV viral particle libraries in organoids, in particular a method for specifically screening for capsids that can specifically infect a population of cells, such as neurons, in preference to other cell types or populations. The present invention also relates to a method for directed evolution by repeating the screening method, AAV viral particles obtained or evolved using the method, and a method for infecting a specific population of cells with the AAV viral particles.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for screening AAV viral particle libraries in organoids, in particular a method for specifically screening for capsids that can specifically infect a population of cells, such as neurons, in preference to other cell types or populations. The present invention also relates to a method for directed evolution by repeating the screening method, AAV viral particles obtained or evolved using the method, and a method for infecting a specific population of cells with the AAV viral particles. [Background technology]

[0002] Background of the Invention Adeno-associated viruses (AAVs) are important vectors for gene therapy. Numerous naturally occurring AAV serotypes have been identified, each of which has differences in the coding sequence of the viral capsid. These differences in the capsid result in altered cell tropism; i.e., different AAV serotypes differentially infect different cell types (Castle et al., 2016).

[0003] Naturally occurring AAV serotypes have been used in both clinical trials and approved gene therapies, but the predominance of antibodies to natural AAV in the human population, coupled with the inability of natural serotypes to efficiently target specific cell types, means that synthetic (non-wild-type) capsids will become increasingly popular as gene medicines ( Kuzmin et al., 2021 ).

[0004] Synthetic AAV capsids have amino acid sequences that are broadly similar to naturally occurring capsids but are constructed in the laboratory via random mutagenesis, DNA shuffling, or other synthetic biology techniques. Synthetic capsids can be rationally designed, for example, by inserting peptide sequences with potentially useful properties. Alternatively, large, diverse libraries of random capsids can be created (Korbelin et al., 2017). Typical libraries contain up to 10 12 A large pool of different variants of the vector can be used to identify those variants with desired properties from this pool, including infection of specific cell types of interest, such as neurons (Bartel et al., 2012), as well as detargeting from other undesired cell types or organs, such as the liver.

[0005] Directed evolution of AAV capsids from libraries can be performed in vivo or in vitro, for example, by injecting the library into an animal and then harvesting the target tissue or cell type (Figure 1A). If desired, this first round of evolution can be supplemented with additional rounds in which the harvested capsid DNA is further mutated or modified and the screening process is repeated (Grim and Bueng, 2017).

[0006] As is generally true for directed evolution approaches, "you get what you screen": the more closely the screening environment replicates the final targeting system, the greater the likelihood of finding candidates that possess the desired properties (Schmidt-Dannert and Arnold, 1999). Therefore, for gene therapy approaches, it is critical that AAV capsid libraries be screened in a system that resembles as closely as possible the eventual human patient.

[0007] Directed evolution of AAV capsids is often performed in laboratory animals such as mice or nonhuman primates (NHPs). Mice are preferred due to their widespread availability and ease of genetic manipulation in experimental investigations, while NHPs are evolutionarily closer to humans. In both instances, capsid libraries are injected into laboratory animals, relevant tissues or cell types are harvested after an incubation period, and the recovered AAV particles are then sequenced to identify useful capsids. As expected, capsid evolution performed in mice yields capsids that are effective in mice but not in NHPs (Ligoure et al., 2019), and capsid properties are also linked to the precise strain of mouse used (Mathieson et al., 2020). As a result, the most effective capsid evolution strategies currently utilize a combination of animal screening and validation in human cells (see Tabeboldbar et al., 2021).

[0008] Organoids, grown from stem and progenitor cells (induced pluripotent stem cells (IPSCs); embryonic stem cells (ESCs)) and composed of organ-specific cell types, can be defined as 3D structures that self-organize through cell sorting and spatially restricted lineages "in a dish" to form representations of specific (human) tissues (Bredenoord et al., 2017). Numerous different types of organoids have been developed for various human tissues, including parts of the gastric system, small intestine, colon, liver, pancreas, trachea, alveoli, thyroid, esophagus, prostate, fallopian tubes, and kidney, as well as various parts of the nervous system, such as the retina, cerebellum, cerebrum, olfactory bulb, hippocampus, hypothalamus, choroid plexus, spinal cord, and neuromuscular system (see Rossi et al., 2018).

[0009] Numerous studies have used organoids to test whether previously identified capsids (either synthetic or wild-type capsids) are effective in human tissues. For example, Achberger et al. (2021) recently used retinal organoids to test the tropism of seven different AAV vectors (both wild-type and synthetic) to assess which had the best tropism, efficacy, and kinetics in human tissues. Similarly, Garita-Hernandez et al. (2020) used retinal organoids to test the efficacy of multiple AAV serotypes in the human retina, and McClements et al. (2022) used retinal organoids to evaluate the effects of both AAV capsids and promoters of gene expression within photoreceptors. Depla et al. (2020) used human brain organoids to test the efficacy of two human-relevant AAV serotypes, AAV5 and AAV9. They found that AAV5 provided superior transduction.

[0010] Outside of the nervous system, human organoids have been used to test the ability of different AAV serotypes to infect the lungs ( Meyer-Berg et al., 2020 ) and to screen six different synthetic capsids for the ability of AAV serotypes to infect cell types in kidney organoids ( Ikeda et al., 2018 ).

[0011] The above studies represent the use of human organoids to test AAV capsids previously identified by other means. Importantly, they substantiate the belief that in vitro human tissue provides a superior environment for testing human therapeutics than animal models. However, no studies or descriptions exist that use human tissue or organoids as a vehicle to investigate and identify novel AAV capsids. Summary of the Invention [Problem to be solved by the invention]

[0012] The present disclosure relates to a method for screening AAV capsids using organoids to select capsids that selectively infect specific cell types, and a method for directed evolution using the organoids, which represents the first step in identifying novel vectors for gene therapy. [Means for solving the problem]

[0013] Summary of the Invention According to a first aspect, there is provided a method for screening for capsid-encoding nucleotide sequences of AAV viral particles capable of infecting population-specific neurons, the method comprising: (i) obtaining or having obtained a neuromuscular organoid comprising neurons and muscle cells, wherein the neurons have a cell body and a distal portion located away from the cell body, and the neurons are arranged within the organoid such that the cell body and the distal portion are distally separated from each other; (ii) contacting the distal portion with a population of AAV viral particles such that the AAV viral particles can infect the neuron at the distal portion of the neuron; (iii) recovering AAV viral particles from the cell body that infected the distal portion of the neuron; and (iv) determining the capsid-encoding nucleotide sequence of AAV viral particles recovered from the cell bodies; Includes.

[0014] In a second aspect, (i) obtaining or having obtained a neuromuscular organoid comprising neurons and muscle cells, wherein the neurons have a cell body and a distal portion located away from the cell body, and the neurons are arranged within the organoid such that the cell body and the distal portion are distally separated from each other; (ii) contacting the distal portion with a population of AAV viral particles such that the AAV viral particles can infect the neuron at the distal portion of the neuron; (iii) recovering from the cell body the AAV viral particles that have infected the distal portion of the neuron; (iv) determining the capsid-encoding nucleotide sequence of the AAV viral particles recovered from the cell bodies; (v) optionally using the output of step (iv) to generate a new AAV viral particle library and repeating the method; A directed evolution method is provided for selecting AAV viral particles capable of selectively infecting population-specific neurons, comprising:

[0015] In one embodiment, the neuromuscular organoids are derived from iPSCs or ESCs.

[0016] In one embodiment, the population-specific neurons are selected from the list consisting of mammalian, human, human subpopulations, and human disease-specific types.

[0017] In one embodiment, the human disease-specific population-specific neurons are selected from the list of MND, DM, ALS, HD, epilepsy, neuropathy, PD, SCA, HSP, PLS, SMA, SBMA and LCCS neurons.

[0018] In a third aspect, AAV viral particles capable of infecting population-specific neurons identified or selectively evolved by the methods disclosed herein are provided.

[0019] In a fourth aspect, there is provided a use of an AAV viral particle according to the present disclosure for selectively infecting population-specific neurons.

[0020] In one embodiment, the AAV viral particle comprises a nucleotide-encoded payload.

[0021] In one embodiment, the nucleotide-encoded payload encodes a therapeutic peptide.

[0022] In one embodiment, the nucleotide-encoded payload is a gene therapy payload.

[0023] In a further aspect, there is provided the use of the AAV viral particles disclosed herein in the treatment of diseases caused by genetic mutations.

[0024] The present invention provides AAV capsids identified by the screening methods of the present invention.

[0025] Advantageously, AAV capsids identified by the screening methods described herein may be used to develop gene therapies for treating various diseases or disorders. Accordingly, the AAV capsids may be used in methods of ameliorating or treating a neuromuscular or neuromotor disease or disorder in a subject, comprising administering to the subject a therapeutically active amount of an AAV expression vector or viral particle of the invention.

[0026] For a better understanding of the present invention, and to show how the same may be carried into effect, specific embodiments, methods and steps according to the present invention will now be described, by way of example only, with reference to the accompanying drawings in which: [Brief explanation of the drawings]

[0027] [Figure 1A] Figure 1A shows a prior method for directed evolution of AAV capsids, in which an AAV genomic capsid library containing a wide range of capsid variants is generated and packaged into an AAV vector capsid library. The library is injected into laboratory animals (19), and the capsids / AAV are then harvested from target tissues (20) and sequenced. [Figure 1B]Figure 1B shows a flowchart of a method for identifying AAV capsid variants from organoids: 1 - generating tissue-specific organoids, 2 - infecting organoids with an AAV capsid library, 3 - harvesting capsid DNA or RNA from the relevant organoid cell type, 5 - sequencing and analysis of enriched variants, and 4 - generating a new library. [Figure 2A] Figure 2 shows an exemplary use of neuromuscular organoids to screen for motor neuron-targeting AAV capsids. Figure 2A shows a spinal cord-neuromuscular organoid with a spatially separated spinal cord compartment (9) containing interneurons (6) and motor neurons (7), and an innervated muscle compartment (10) containing motor neuron axons and synaptic terminals (8). [Figure 2B] Figure 2 shows an exemplary use of neuromuscular organoids to screen for motor neuron-targeting AAV capsids. Figure 2B shows the microinjection (11) of an AAV capsid library (12) into the muscle compartment, where some library members infect motor neuron synaptic terminals and are retrogradely transported from within the motor neuron axon (13) to the motor neuron cell body (14) in the spinal cord compartment, resulting in the release of capsid DNA. [Figure 2C] Figure 2 shows an exemplary use of neuromuscular organoids to screen for motor neuron-targeting AAV capsids. Figure 2C shows the microdissection and harvesting of a motor neuron (7) and dissociation of the cell body (16) from the rest of the tissue. [Figure 2D] Figure 2 shows an exemplary use of neuromuscular organoids to screen for motor neuron-targeting AAV capsids. The cells in Figure 2D are lysed (17) to release capsid DNA, which is then sequenced (18) to identify capsids that infected motor neurons. These capsids can then be further validated and subjected to additional rounds of evolution. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description As used herein, screening refers to a method for selectively identifying the members of a population for a desired property.In this specification, the population is an AAV library or an AAV virus particle library, and the desired property is the ability to selectively infect population-specific cells, such as population-specific neurons.

[0029] As used herein, a capsid-encoding nucleotide sequence refers to a nucleotide sequence that encodes a capsid protein. Typically, the nucleotide sequence is a DNA sequence, although RNA, modified nucleotides, and synthetic nucleotides are also contemplated.

[0030] As used herein, AAV virus particle refers to the virus particle of adeno-associated virus, which comprises a single-stranded DNA genome packaged in a viral envelope and can infect cells, such as mammalian cells.In particular, as used herein, AAV virus particle comprises the nucleotide sequence for the capsid that packages nucleotide sequence.In some embodiments, virus particle can lack the nucleotide that codes for replication, that is, they are replication-deficient.

[0031] AAV is a small virus of approximately 4.9 kb in size belonging to the Dependoparvovirus genus, containing a single strand of DNA. The AAV genome contains three capsid proteins, VP1, VP2, and VP3, all of which are translated from a single mRNA via alternative splicing. Multiple wild-type AAV serotypes have been identified, each with a unique arrangement of capsid genes and distinct tropisms, but wild-type serotypes tend to be capable of infecting multiple tissues and cell types. These serotypes are designated by numbers, such as AAV1, AAV2, and others. Modification of capsid sequences via recombinant DNA techniques has been shown to generate non-native sequences that penetrate the immune system with tailored properties and tropisms to target (or repel) specific cells or tissues (Vandenberghe et al., 2009).

[0032] Modifications to capsids can be achieved in several ways, including random mutagenesis of existing capsid DNA sequences, capsid shuffling (i.e., taking DNA sequences from multiple capsids and randomly shuffling portions of the sequences to create new capsids; Buning et al., 2015), or by inserting short peptide sequences into exposed loop regions of the capsid. These methods can generate a large number of highly diverse capsids with potentially valuable properties. These can be screened in animal tissues or cell cultures to select those capsids that, once packaged into functional virions, target specific tissues or cells. These capsid sequences can then be generated de novo and combined with genes of therapeutic potential in gene therapy.

[0033] As used herein, "capable of infecting population-specific neurons" refers to the ability to selectively infect a particular cell type, such as neurons rather than muscle tissue.

[0034] However, population specificity may also refer to selectively infecting animal or human neurons, for example, human neurons rather than rat neurons.

[0035] Additionally, the methods described herein may be used to identify viral particles capable of infecting subpopulations of neurons, such as human neurons in diseased rather than non-diseased states, including Duchenne muscular dystrophy (DM), amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease (HD), neuropathy, Parkinson's disease (PD), spinocerebellar atrophy (SCA), hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), and lethal congenital contracture syndrome (LCCS), or other genetic disorders.

[0036] Furthermore, population specificity may relate to the ability to selectively infect parts of neurons, such as axons or dendrites rather than the cell body.

[0037] As used herein, the term neuron encompasses neurons and portions or portions thereof (e.g., the cell body, axon, or dendrites of a neuron). As used herein, the term neuron refers to a nervous system cell that includes a central cell body (or soma) and two types of extensions or processes: dendrites, which carry most of the neuronal signal to the cell body, and axons, which carry most of the neuronal signal from the cell body to target neurons or effector cells such as muscles. Neurons can carry information from tissues and organs to the central nervous system (afferent or sensory neurons) and can transmit signals from the central nervous system to effector cells (efferent or motor neurons). Other neurons, called interneurons, connect neurons within the central nervous system (brain and spinal cord). Still other neurons, called projection (or "projection") neurons, extend axons from one region of the nervous system to another. The screening methods disclosed herein may be used to screen for capsid-encoding nucleotide sequences of adeno-associated virus ("AAV") particles capable of infecting such neurons.

[0038] It will be appreciated that the methods disclosed herein may be applicable to cell types other than neurons.

[0039] In some embodiments, the population-specific neurons are motor neurons.

[0040] In other embodiments, the population-specific neurons are sensory neurons.

[0041] In some embodiments, the population-specific neurons are interneurons (or genetically defined subtypes).

[0042] In some embodiments, the population-specific neurons are projection neurons (or genetically defined subtypes).

[0043] In one embodiment, the population-specific neurons are human neurons.

[0044] In one embodiment, the population-specific neurons are human neurons in a disease state.

[0045] As used herein, a neuron in a disease state refers to a neuron or neuron subtype involved in the pathophysiology of a particular disease, such as dopaminergic neurons in Parkinson's disease or motor neurons in amyotrophic lateral sclerosis, etc.

[0046] As used herein, organoid refers to a miniaturized and simplified version of an organ generated in vitro in three dimensions, showing realistic microanatomy. They are derived from one or several cells of tissue, embryonic stem cells, or induced pluripotent stem cells, and can self-organize in three-dimensional culture due to their self-renewal and differentiation capabilities. In the example of neuromuscular organoids, they are composed of both muscle and neurons, and may contain both motor neurons and spinal cord. Neuromuscular organoids are described, for example, in Martins et al. (2020), Periera et al. (2021), and Andersen et al. (2020). Other types of nervous system organoids, such as retina, cerebellum, cerebrum, spinal cord, etc., are envisioned in the present invention.

[0047] In one embodiment, the organoids are derived from iPSCs or ESCs.

[0048] In one embodiment, the organoids are human organoids.

[0049] In one embodiment, the organoids are organoids in a human disease state.

[0050] In one embodiment, the organoids are neuromuscular organoids.

[0051] As used herein, a disease state organoid is an organoid grown from a cell or tissue known to carry a genetic mutation that results in a pathology.

[0052] Distant separation as used herein means that neuronal part is located at the physically separate part of organoid, so that viral particle can be preferentially delivered to neuronal part such as axon or dendrite, and other part such as cell body.Advantageously, by only collecting the viral particle found in, for example, cell body or spinal cord, it can determine which particle can infect neuron.

[0053] Distal portion as used herein refers to the portion of the neuron that is distal to the cell body, such as the axon and dendrites, especially the axon.

[0054] Without wishing to be bound by theory, it is thought that some AAV particles may target receptors specifically located in axons and use these receptors to infect neurons.

[0055] As used herein, " contacting the distal part of neuron with a group of viral particles, so that viral particles can infect neuron at the distal part of neuron " refers to any method that exposes the distal part of neuron to viral particles.This can include but is not limited to injection into specific compartment of organoid (such as the muscle compartment of neuromuscular organoid), microinjection, or contain AAV vector in culture medium.

[0056] As used herein, "recovering viral particles that have infected the distal portion of neurons from the cell bodies" refers to any method that selectively recovers only viral particles that have successfully infected neurons by harvesting the cell bodies. This can include, but is not limited to, harvesting components of the culture, such as microdissection of the spinal cord compartment within neuromuscular organoids, or somatic inclusion of a fluorophore in the target cell type, followed by dissociation of the organoids and harvesting the target cell type via fluorescence-activated cell sorting (FACS). As used herein, "determining the capsid-encoding nucleotide sequence of the viral particles recovered from the cell bodies" refers to sequencing the viral particle nucleotide sequence of those viral particles recovered from the cell bodies, typically using high-throughput sequencing of a type known in the art, such as next-generation sequencing (NGS).

[0057] Advantageously, the nucleotide sequence of the virus particle collected from cell body can be compared with the sequence of the whole library or other source, for example, the nucleotide sequence collected from the muscle cell of neuromuscular organoid or other types of neuron.By comparing the difference between these sequences, can identify trends, and can rationally design additional AAV library.

[0058] Advantageously, the viral nucleotide sequences can then be analyzed and used to generate new libraries that can be used in the screening methods described herein that provide for novel directed evolution methods.

[0059] In vivo studies have shown that directed evolution of AAV capsids can lead to vectors with useful properties, such as the ability to cross the blood-brain barrier and neurotropism (Deverman et al., 2016 and EP 3044318), or targeting dopaminergic neurons (Daviddson et al., 2019) or cardiomyocytes (Yang et al., 2009), among others (see Li and Sumulski, 2020 for a review). Examples of directed evolution include the creation of capsid libraries, mixtures of AAV vectors encapsidated with random capsid sequences generated via error-prone PCR, capsid shuffling, or other methods. These libraries have been applied to cell lines (e.g., Maheshri et al., 2006), undifferentiated stem cells (Asuri et al., 2012), or most commonly, experimental animals (e.g., Devermann et al., 2016; Li and Sumulski, 2020; U.S. Patent No. 8,632,764 B2; U.S. Patent No. 20170166926 A1; U.S. Patent No. 9,701,984 B2). The present invention advantageously provides a method for directed evolution using organoids.

[0060] As used herein, population-specific AAV viral particles refer to AAV viral particles that selectively infect population-specific neurons, such as AAV viral particles identified or evolved using the methods disclosed herein.

[0061] As discussed above, AAV capsids have the potential for impressive levels of cell and species tropism, but currently available screening techniques are unable to fully exploit this selectivity and utilize it for therapeutic benefit.

[0062] The present invention overcomes these drawbacks and enables screening of AAV capsid libraries in organoids derived from induced pluripotent stem cells ("iPSCs") or embryonic stem cells ("ESCs"). Organoids can be derived in vitro from iPSCs or ESCs (Little et al., 2019). Because the genetic makeup of these organoids is more similar to that of human patients than the same cells found in animal models, they are believed to provide a much more suitable substrate for screening AAV libraries than experimental animals. In some embodiments, the organoids are neuromuscular and the neurons are motor neurons.

[0063] The present invention also includes a virus evolution approach that utilizes a combination of artificial iPSC and ESC-derived organoids and in vitro screening to identify the AAV capsid-encoding nucleotide sequence that allows AAV capsids to more efficiently infect neurons, particularly population-specific neurons.In some embodiments, this approach allows the identification of the AAV capsid-encoding nucleotide sequence that allows AAV capsids to more efficiently infect motor neurons via intramuscular injection.

[0064] The present invention also includes a technology platform that uses iPSC / ESC-derived organoids to identify novel adeno-associated virus (AAV) capsids based on their ability to target population-specific neurons. An exemplary use of this is described herein, where an AAV capsid library can be screened for its ability to efficiently infect population-specific neuronal terminals, but the same system can be used to identify capsids that target multiple neuronal types.

[0065] Furthermore, because the capsid sequences that efficiently infect neurons can vary from subject to subject, the present invention further includes a step in which skin samples from individual subjects can be removed and transformed into stem cells and then into organoids, and these organoids from patients can be used to screen for effective AAV vectors, providing a personalized approach to gene therapy.

[0066] In some examples, iPSCs or ESCs are derived from a subject. In some examples, iPSCs or ESCs are derived from a subject's skin sample. In some examples, iPSCs or ESCs are derived from a subject's fibroblasts. In some examples, the subject is a human subject. iPSCs and ESCs can also be obtained from animals, human subjects / patients, or cell banks. Screening an AAV capsid library using iPSCs from a human subject or patient allows for the identification of capsid sequences that infect neurons or other cells from the subject or patient. Thus, capsid sequences can be produced on an individualized basis using this method.

[0067] In some examples, the method is a method of screening for capsid-encoding nucleotide sequences of adeno-associated virus ("AAV") particles capable of infecting neurons of a subject via intramuscular injection.

[0068] In one embodiment, the organoids are derived from iPSCs.

[0069] In one embodiment, the organoids are derived from ESCs.

[0070] In some embodiments, the iPSCs used in the screening methods described herein are derived from a human subject.

[0071] In some embodiments, iPSCs are derived from a culture of fibroblasts from a skin biopsy. Several methods that allow for the culture of fibroblasts from a skin biopsy have been previously described (Vangipuram M, Ting D, Kim S, Diaz R, Schule B. Skin punch biopsy explant culture for derivation of primary human fibroblasts. J Vis Exp. 2013;(77):e3779. Published July 7, 2013. doi:10.3791 / 3779).

[0072] In some embodiments, the iPSCs used in the screening methods described herein are derived from a human subject.

[0073] In some embodiments, the iPSCs are derived from a culture of fibroblasts from a skin biopsy of a subject.

[0074] Derivation of human fibroblasts into iPSCs can be achieved using commercially available kits, such as the CytoTune-IPS Sendai Reprogramming Kit from ThermoFisher. Further details can be found at https: / / www.thermofisher.com / order / cataloq / product / A16517# / A16517 and https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / cvtotune ips 20 sendai reproq kit man.pdf.

[0075] The recombinant AAV (rAAV) vectors developed using the methods described herein are particularly applicable to the treatment of neuromuscular / neuromotor disorders such as spasticity, amyotrophic lateral sclerosis, and dystonia, allowing for the introduction of genetic material into motor neurons via intramuscular injection of viral vectors. However, the screening methods defined herein are independent of the disease being treated and may be used to screen for capsid sequences that effectively target many neuronal types, and thus may be effective in gene therapy approaches regardless of the neuronal disorder (or "neuronal disorder"). It is believed that the present invention may also be applied to other types of neurons underlying neurological disorders, as well as other neuronal disorders that currently have few or no effective treatments.

[0076] The efficacy of AAV particles to infect population-specific neurons can be determined by counting the number of neurons expressing viral DNA.

[0077] For example, multiple motor neurons innervate the same target, for example, a muscle in the case of motor neurons, and the proportion of these neurons transduced by AAV viral particles can be counted. The effectiveness of AAV viral particles in infecting neurons or specific parts of neurons can also be determined by DNA sequencing or RT-PCR, which focuses on the "copy number" of viral DNA present in neuronal cells. This provides an estimate of the number of times the same cell is infected with AAV viral particles.

[0078] Application of an AAV capsid library to distal parts of neurons (such as axons, dendrites, neurites, or the "synaptic terminals" of axon terminal portions) and harvesting genetic material from another region of the cell (such as the cell body) allows for the identification and determination of AAV capsid sequences that successfully infect those cells via specific pathways.

[0079] It is contemplated that the screening methods described herein can be applied to screen for capsid-encoding nucleotide sequences of AAV viral particles capable of infecting other cell types, such as sensory neurons, basal ganglia projection neurons, dopaminergic neurons, and muscle tissue. Advantageously, this would allow the identification of capsid sequences that infect other cell types, allowing capsids to be designed away from those sequences, thereby reducing the likelihood of cross-infection.

[0080] In some examples, the screening methods described herein include providing, for example, by direct intramuscular injection, a plurality of test AAV viral particles that are additionally screened for their ability to infect muscle cells. The additional screening may occur before, after, or simultaneously with screening of particles for their ability to infect neurons. Advantageously, this type of comparative data allows for the selection of capsids that target desired cell types.

[0081] In some cases, this screening method allows the use of multiple organoids in a single culture system.For example, organ chips or organoid fusion technology (Kakni et al., 2022) can be used to maintain multiple organ systems in one in vitro system.For example, dorsal and ventral forebrain organoids can be fused to generate interacting spheroids (Birey et al., 2017); assembled thalamic and cortical organoids contain reciprocal thalamocortical and corticothalamic organoids (Xiang et al., 2019); and multiple organ systems, such as liver, heart and lung, can be integrated into a single chip to study the interaction across multiple systems, in this example, to study AAV capsid infectivity (Skardal et al., 2017).The use of multiple organoids in a single culture system provides a screening environment that is more similar to a complete human system than targeting one organ or cell type over others, further improving the ability to identify AAV capsids.

[0082] The screening method described herein requires the creation of an AAV viral particle library or capsid library. Methods for creating libraries are known in the art. One such method is described in Nonenmacher et al., 2021.

[0083] Diverse capsid libraries can be generated, for example, through the following processes: i) random mutagenesis of naturally occurring capsids; ii) shuffling of naturally occurring capsids; iii) insertion of targeted or random peptide sequences up to 25 amino acids in length in various regions within VP1, VP2, or VP3 of the AAV capsid; or iv) combinations of the above.

[0084] To generate the library, random capsid sequences are cloned into an AAV backbone containing AAV2 inverted terminal repeats (ITRs; packaging signals) and the AAV rep gene. These DNA plasmids are transfected into HEK293 cells in the presence of additional adenovirus genes to facilitate AAV packaging. AAV virions are harvested, purified, and concentrated from HEK293 cells and / or culture medium according to standard methods (e.g., Potter et al., 2014 https: / / dx.doi.ora / 10.1038%2Fmtm.2014.34; McClure et al., 2011 htp: / / dx.doi.Org / 10.3791 / 3348).

[0085] In some embodiments, the method for generation of an AAV library comprises one or more of the following steps: - The purified and enriched AAV library is diluted in Dulbecco's Modified Eagle Medium (DMEM) and applied to the muscle chamber of the microfluidic device. - 2-10 days after application, neuronal cell bodies are harvested either by chemical (i.e. trypsinization) or mechanical (cell detachment) methods. Neurons can be lysed and the lysates subjected to deep sequencing (such as RNA or DNA sequencing) to directly detect capsid sequences found within the neurons, or the lysates can be used as PCR templates with primers directed against conserved regions of the AAV capsid. Following PCR of the capsid region, DNA fragments are cloned into DNA vectors and subjected to Sanger sequencing. Capsid sequences collected from neurons are analyzed bioinformatically for conserved regions, and highly enriched capsids can be synthesized de novo and then subjected to either further mutagenesis or repeated in vitro screening to increase evolutionary pressure through directed evolution. - Directed evolution can be repeated for multiple rounds (approximately 2-5 rounds). Capsid sequences that demonstrate efficient retrograde transport in vitro can be used to generate functional virions for in vivo use.

[0086] In one embodiment, viral particles that selectively infect population-specific neurons (i.e., population-specific AAV viral particles) are used to deliver a nucleotide-encoding payload or exogenous transgene to infected population-specific neurons, which can advantageously be used to deliver gene therapy to target cells.

[0087] In one embodiment, population-specific AAV viral particles can be used as viral vectors, such as expression vectors, which can be used in gene therapy to deliver exogenous transgenes to subjects in need thereof.

[0088] Typically, gene therapy will be used to treat neurological disorders or diseases. One example of a neurological disorder or disease that can be treated by gene therapy is spasticity. Spasticity is a neurological symptom afflicted by people with various neurological disorders, including but not limited to multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, and cerebral palsy. Spasticity results from excessive excitation of muscles by motor neurons, which become "hyperexcitable" due to this disease.

[0089] The present invention also includes methods of treatment that involve injecting AAVs containing novel population-specific AAV capsids identified using the screening or directed evolution methods disclosed herein into affected muscles of a subject; these AAVs can then infect distal portions of motor neurons and be transported to their cell bodies, leading to the expression of exogenous transgenes in population-specific neurons that specifically innervate that muscle, providing high specificity.

[0090] The present invention therefore enables the creation of viral vectors (or expression vectors) that access population-specific neurons and then modify gene expression in population-specific neurons, with the goal of curing, alleviating symptoms, and / or improving the quality of life of patients with diseases affecting, for example, motor neurons.

[0091] The present invention also includes methods of treatment comprising injecting AAV viral vectors containing novel population-specific AAV capsids into the brain or spinal cord of a subject, which AAV viral vectors can then infect distal portions of the subject's motor neurons, sensory neurons, interneurons, or projection neurons, and may selectively infect distal portions of those neurons in specific populations.

[0092] The present invention therefore enables the creation of viral vectors that access these neurons following intracranial, intraspinal or intramuscular injection and then modify gene expression in these neurons with the goal of curing, alleviating symptoms and / or improving the quality of life of patients with diseases that affect these neurons.

[0093] The capsid nucleotides identified by the methods of the present invention may be used to develop gene therapies that include viral vectors that access neurons after intramuscular injection and then modify activity and / or gene expression within the neurons. The nucleotides may also be used to develop gene therapies that include viral vectors that access neurons after intramuscular injection and then modify activity and / or gene expression within the neurons.

[0094] In some embodiments, the viral vector is capable of modifying the activity of targeted population-specific neurons in a subject. In some embodiments, the viral vector is capable of modifying the activity of targeted motor neurons in a subject via intramuscular injection.

[0095] In some embodiments, the viral vector further comprises a transgene encoding a transgene product, wherein the transgene product is capable of modifying the activity of targeted population-specific neurons in a subject. In some embodiments, the transgene product is capable of modifying the activity of targeted population-specific neurons in a subject via intramuscular injection.

[0096] In some embodiments, the method for the addition of a payload that modifies neuronal activity may include one or more of the following steps: - Capsid sequences identified by the screening methods described herein are synthesized de novo and inserted into an AAV helper plasmid containing the AAV2 REP genes (Rep / Cap). - The Rep / Cap plasmid is combined with an additional plasmid containing the AAV backbone and adenovirus helper genes (such as pHelper) and transiently transfected into HEK293 cells. AAV particles can be purified using standard methods and used in in vitro or in vivo experiments (e.g., Potter et al., 2014 https: / / dx.doi.org / 10.1038%2Fmtm.2014.34: McClure et al., 2011 http: / / dx.doi.org / 10.3791 / 3348).

[0097] The present invention also provides AAV viral particles, eg, AAV viral particles produced by the methods described herein.

[0098] In some embodiments, the AAV capsid is "individualized" to the subject, and the method of treatment will be specific (personalized) to the subject being treated by creating personalized organoids.

[0099] In some embodiments, the methods involve AAV expression vectors or viral particles retrogradely infecting neurons to deliver genetic material to the neurons for the purpose of treating a neuromuscular or neuromotor disorder, or a disorder affecting movement, or any disorder that targets neurons for therapeutic purposes.

[0100] In some embodiments, AAV expression vectors or viral particles are delivered intramuscularly to retrogradely infect motor neurons of a subject to alter motor neuron activity in the subject.

[0101] As used herein, "retrograde transport" or "retrograde infection" refers to uptake of a vector at the axon terminal (or "synaptic terminal"), i.e., at the synapse, and transport down the axon in the direction opposite to the direction of action potential propagation (hence "retrograde") to the cell body of the neuron. The viral nucleic acid can then enter the nucleus, where it can replicate and become transcriptionally and translationally active.

[0102] Such delivery is advantageous when the neuronal cell body and / or axon itself is inaccessible, but terminal projection fields, including synapses, are available for gene vector delivery. Thus, successful delivery of a retrogradely transportable gene vector to such terminal projection fields results in retrograde transport and infection of vulnerable projection neurons.

[0103] When the viral vector is delivered to the neuronal cell body, the viral nucleic acid typically localizes to the nucleus of the cell. According to some embodiments of the present invention, adeno-associated viral particles that undergo retrograde delivery to the neuronal cell body can insert their nucleic acid contents directly into the nucleus.

[0104] Embodiments of the present invention include the delivery of substantially non-toxic recombinant adeno-associated viral vectors carrying a heterologous gene of interest to provide retrograde gene delivery to neuronal cell bodies resulting in gene expression.

[0105] In some embodiments, the AAV expression vectors of the invention are able to access motor neurons after intramuscular injection and then modify gene expression in motor neurons to treat neuromuscular or neuromotor disorders, leading to a cure, relief of symptoms, and / or improved quality of life for patients with diseases affecting motor neurons.

[0106] As used herein, a "neurological disorder" refers to a disorder that causes morphological and / or functional abnormalities in nerve cells or a population of nerve cells. A neurological disorder can result in the impairment or absence of normal nerve function in a subject, or the presence of abnormal nerve function. For example, a neurological disorder can be the result of disease, injury, and / or aging. Non-limiting examples of morphological and functional abnormalities include physical deterioration and / or death of nerve cells, abnormal growth patterns of nerve cells, abnormal physical connections between nerve cells, under- or over-production of one or more substances by nerve cells, such as neurotransmitters, inability of nerve cells to produce one or more substances that they normally produce, production of substances, such as neurotransmitters, and / or generation or transmission of electrical impulses in an abnormal pattern or at an abnormal time. As used herein, a "neuromotor disorder" refers to a developmental or acquired disorder that typically affects movement / gross motor disorders, posture, and fine motor disorders. The disorder is caused by damage to the central nervous system. It can be due to developmental problems in the cortex, basal ganglia, thalamus, cerebellum, brainstem, spinal cord, or peripheral nerves, or damage to their developing motor pathways. The most common neuromotor disorders in children include cerebral palsy, muscular dystrophy, and spina bifida. The most common neuromotor disorders in adults include stroke, multiple sclerosis, Parkinson's disease, and traumatic injury. Injury can be static (non-regressing) or progressive.

[0107] The term "treatment" also includes combination treatments and therapies in which two or more treatments or therapies are combined, for example sequentially or simultaneously.

[0108] The present invention also provides a cell comprising an AAV viral vector described herein. In some embodiments, the cell is a mammalian cell, such as a human cell.

[0109] In the context of this specification, "including" should be interpreted as "including."

[0110] Aspects of the invention comprising particular elements are intended to extend to alternative embodiments that "consist" or "consist essentially of" the associated elements.

[0111] Where technically appropriate, embodiments of the present invention may be combined.

[0112] Embodiments are described herein as comprising particular features / elements. The disclosure extends to other embodiments that consist of, or consist essentially of, said features / elements.

[0113] Technical references, such as patents and applications, are incorporated herein by reference.

[0114] Any embodiment specifically and explicitly recited herein may form the basis for a disclaimer, either alone or in combination with one or more additional embodiments.

[0115] Example Example 1 – AAV capsid screening in organoids grown in microfluidic chambers 1. hiPS Cell Culture and Differentiation a. iPS cells were cultured on vitronictin-coated dishes with Essential 8 medium to maintain an undifferentiated state. b. hiPSCs were dissociated with Accutase and plated onto Matrigel-coated dishes in Essential 8 medium containing ROCK inhibitor and cultured until the cells reached 80% confluence. The hiPSCs were then dissociated with Accutase, resuspended in differentiation medium N2B27, and cultured on a non-adherent, ultra-low attachment surface. c On day 2, embryoid bodies were formed, transferred to new dishes, and cultured in N2B27 containing Chir-99021. d. On day 4, retinoic acid and smoothon agonist were added. e. On day 9, DAPT was added. f. From day 11 onwards, BDNF and GDNF were added to the differentiation medium, and the medium was changed every other day.

[0116] 2. Formation of Motor Neuron Organoids a. Differentiated cells were dissociated with Accutase and seeded into low-adhesion V-bottom 96-well plates for 10 days to generate spheroids. b. A microfluidic device was coated with Matrigel, and spheroids were transferred to one side of the microfluidic chamber. Half of the culture medium was replaced with fresh medium every 4 days. c. hIPSC-derived myoblasts were seeded on the other side of the microfluidic chamber. d. Typically 20-30 days after the spheroids are transferred to the microfluidic chamber, axon bundles form within the microgrooves and connect with myocytes growing in the other channel.

[0117] 3. Addition of AAV Capsid Library a. The library was added to the side where muscle cells were growing and forming connections with axon terminals. b. To ensure fluid isolation between both chamber sides, 8 hours after the library was added, the medium was exchanged to the muscle side.

[0118] 4. Collection of Neural Organoids and Rescued Capsid Sequences by PCR a. After 7 days, neural organoids were dissected by manually cutting open the microfluidic device with a razor blade. b. After harvesting the spheroids, DNA was extracted from the spheroids using a miniprep DNA kit. c. Capsid sequences administered to infect neural organoids were recovered by PCR using specific capsid primers. d. PCR products were cloned back into the library plasmid and packaged into AAV. e. Capsid sequences collected from neurons are analyzed using bioinformatics for conserved regions, and highly enriched capsids can be synthesized de novo and then subjected to either further mutagenesis or repeated in vitro screening to increase evolutionary pressure through directed evolution. f. Directed evolution can be repeated for multiple rounds (about 2-5 rounds).

[0119] Example 2 – AAV capsid screening via direct injection of an AAV library into the organoid compartment 1. hiPS Cell Culture and Differentiation a, hiPS cells were cultured on vitronictin-coated dishes with Essential 8 medium to maintain an undifferentiated state. b. hiPS cells were grown for at least three passages, and after they reached 70% confluence, they were dissociated into single cells using Accutase. Single cells were plated onto Geltrex-coated p35 dishes. c. On day 1, cells were plated in Neurobasal medium supplemented with Rock inhibitor, CHIR99021, and bFGF. The next day, Rock inhibitor was removed, and cells were maintained in NB medium supplemented with 3 μM CHIR99021 and bFGF until day 3. Medium was changed daily.

[0120] 2. Creation of neuromuscular organoids in 3D a. Neuromesodermal precursors generated from human PSCs in previous steps were dissociated using Accutase to generate single cell suspensions. b. On day 0 of organoid formation, NMP cells were seeded into ultra-low binding 96-well plates containing Neurobasal medium with Rho-associated protein kinase ROCK inhibitor, bFGF and IGF1 and HGF. c. After 4 days, organoids were maintained in NB medium without added growth factors. d. On day 10, organoids were transferred to a 60 mm dish containing 5 mL of medium, and one month later, to a 100 mm dish containing 12 mL of NB medium. During the entire period, organoids were maintained on an orbital shaker rotating at 75 rpm.

[0121] 3. Addition of AAV Capsid Library a. AAV libraries were microinjected into the muscle side of neuromuscular organoids. b. Organoids were maintained on an orbital shaker for 7 days.

[0122] 2. Collection of neural organoids and PCR-rescued capsid sequences a. After 7 days, neural organoids were microdissected by manually cutting the organoids open with a microscalpel. b. After collecting the neuronal side, DNA was extracted from the neuronal side using a miniprep DNA kit. c. Capsid sequences administered to infect neural organoids were recovered by PCR using specific capsid primers. d. PCR products were cloned back into the library plasmid and packaged into AAV. e. Capsid sequences collected from neurons are analyzed using bioinformatics for conserved regions, and highly enriched capsids can be synthesized de novo and then subjected to either further mutagenesis or repeated in vitro screening to increase evolutionary pressure through directed evolution. f. Directed evolution can be repeated for multiple rounds (about 2-5 rounds).

Claims

1. 1. A method for screening for a capsid-encoding nucleotide sequence of an AAV viral particle capable of infecting population-specific neurons, said method comprising: (i) obtaining or having obtained neuromuscular organoids comprising neurons and muscle cells, wherein the neurons have a cell body and a distal portion located away from the cell body, and the neurons are arranged within the organoid such that the cell body and the distal portion are distally separated from each other; (ii) contacting the distal portion with a population of AAV viral particles such that the AAV viral particles are capable of infecting the neuron at the distal portion of the neuron; (iii) recovering from the cell body the AAV viral particles that infected the distal portion of the neuron; and (iv) determining the capsid-encoding nucleotide sequence of the AAV viral particles recovered from the cell bodies; A method comprising:

2. 1. A directed evolution method for selecting AAV viral particles capable of selectively infecting population-specific neurons, comprising: (i) obtaining or having obtained a neuromuscular organoid comprising neurons and muscle cells, wherein the neurons have a cell body and a distal portion located away from the cell body, and the neurons are arranged within the organoid such that the cell body and the distal portion are distally separated from each other; (ii) contacting the distal portion with a population of AAV viral particles such that the AAV viral particles can infect the neuron at the distal portion of the neuron; (iii) recovering from the cell body the AAV viral particles that infected the distal portion of the neuron; (iv) determining the capsid-encoding nucleotide sequence of the AAV viral particles recovered from the cell bodies; (v) optionally using the output of step (iv) to generate a new AAV viral particle library and repeating the method; A method comprising:

3. 3. The method of claim 1 or 2, wherein the neuromuscular organoid is derived from iPSCs or ESCs.

4. The method of any of claims 1 to 3, wherein the population-specific neurons are selected from the list consisting of mammalian, human, human subpopulations, and human disease-specific types.

5. 5. The method of claim 4, wherein the human disease-specific population-specific neurons are selected from the list of MND, DM, ALS, HD, epilepsy, neuropathy, PD, SCA, HSP, PLS, SMA, SBMA and LCCS neurons.

6. An AAV viral particle capable of infecting population-specific neurons identified or selectively evolved by the method of any one of claims 1 to 5.

7. Use of the AAV viral particles of claim 6 for selectively infecting population-specific neurons.

8. The use of claim 7, wherein the AAV viral particle comprises a nucleotide-encoded payload.

9. The use of claim 8, wherein the nucleotide-encoded payload encodes a therapeutic peptide.

10. 10. The use of claim 8 or claim 9, wherein the nucleotide-encoded payload is a gene therapy payload.

11. Use of the AAV virus particle of claim 6 in the treatment of a disease caused by a genetic mutation.

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