method

Abstract

The present invention provides gene or RNA therapeutic vectors for use in the treatment of non-malignant CNS disorders, which are to be injected into the pons by convection-enhanced delivery (CED). The invention also provides a method for treating non-malignant CNS disorders, comprising injecting a gene or RNA therapeutic vector into the pons of a subject in need (CED), and a method for distributing a gene therapy agent throughout the nervous system, wherein a viral vector transported anterograde and retrogradely is injected into the pons and middle cerebellar peduncle of a subject from a cannula inserted from the cerebellum and middle cerebellar peduncle into the pons.

Description

[Technical Field]

[0001] The present invention relates to a gene or RNA therapy vector for use in the treatment of non-malignant CNS disorders, which is administered by convection-enhanced delivery (CED). [Background technology]

[0002] Gene and RNA therapies are being developed for a wide range of neurological disorders affecting the motor and sensory systems. However, delivering these therapies to clinically relevant targets in the motor and sensory systems is extremely challenging due to the complex distribution of corresponding subsystems throughout the central nervous system (CNS).

[0003] The control of motor function (movement) requires the complex and multifaceted coordination of neural signals from interconnected regions of the CNS, including the cerebral cortex (primary motor cortex, premotor cortex, and supplementary motor area), motor thalamus, basal ganglia, brainstem, cerebellum, descending (corticospinal) tract of the spinal cord, and ultimately the peripheral nervous system. Continuous feedback, modification, and fine-tuning processes also arise from inputs from the sensory components of the CNS, including peripheral nerves, dorsal root ganglia, ascending (spinothalamic) tract of the spinal cord, sensory thalamus, and sensory cortex. Furthermore, interconnections between motor and sensory pathways exist at all levels. Inputs from the visual and auditory systems are also necessary for modifying and adjusting motor control.

[0004] Motor and sensory functions are continuously modulated by the limbic system, which includes structures such as the temporal lobe, amygdala, and hippocampus, which play crucial roles in emotional and social processing, motivation, memory, and learning.

[0005] The sensory system is crucial for motor control. The influence of the sensory system is complex and highly integrated, requiring coordination of inputs from the visual, auditory, vestibular, and somatosensory components of the nervous system. A fundamental example of the neural circuitry for motor control is that which supports proprioception (the unconscious understanding of limb position in space). Components of this circuit include muscle spindles, ascending tracts within Clark's column, the posterior column, the gracilis and cuneate nuclei, the medial lemniscus, the sensory thalamus, and the sensory cortex.

[0006] A wide range of neurodegenerative conditions affect multiple components of the nervous system that underpin motor control and sensorimotor integration. Genetic and RNA therapies have the potential to reverse or slow the progression of numerous neurological diseases if their vectors can be delivered to the affected CNS tissue at effective concentrations. In many neurological diseases, pathological changes affect the widely distributed motor areas of the brain, brainstem, cerebellum, and spinal cord, as well as the sensory components of the CNS, including sensory nuclei and sensory pathways. For example, generalized paresthesia is associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder thought to be primarily a disease of motor neurons.

[0007] A significant limitation in the treatment of neurological disorders is the blood-brain barrier (BBB), which prevents many systemically administered therapeutic agents, including viral vector-mediated gene therapies, from reaching effective concentrations in the central nervous system (CNS). Numerous drug delivery strategies have been employed to deliver gene therapies, particularly adeno-associated virus (AAV)-based vectors, to treat neurological disorders characterized by degeneration of motor and / or sensory components of the CNS. Systemic delivery, intra-CSF (intraspinal fluid and cisterna magna) delivery, and intraparenchymal delivery have been investigated. However, all of these strategies have limitations and risks that undermine the ultimate goal of safely, accurately, and effectively achieving therapeutic transgene expression within interconnected neuroanatomical targets supporting motor-sensory integration. These strategies and their drawbacks include:

[0008] Systemic (intravenous) administration: For example, Zolgenesma, a drug used to treat spinal muscular atrophy. - High doses and large volumes of vectors are required, which is costly. - Due to poor penetration into the CNS, its effectiveness is limited (less than 1% of the dose of most systemically administered drugs reaches the brain (Kimura & Harashima, 2020)). - The CNS is targeted nonspecifically. - Associated with systemic toxicity, including hepatotoxicity. - Neutralizing antibodies in circulation may counteract the effect.

[0009] Intra-CSF delivery intrathecal delivery For example, AAV9 / JeT-GAN for giant axonal atrophy (Bailey et al., 2018). - High doses and large volumes of vectors are required, which is costly. - CNS penetration is concentration-dependent, resulting in untargeted, poor, and uneven CNS penetration. - Locally elevated concentrations may lead to inflammation and damage to the spinal dorsal root ganglia and sensory pathways. - It is rapidly absorbed into the systemic circulation. - Effective dosages for the motor cortex and subcortical motor nuclei have not been demonstrated.

[0010] Intracisternal (ICM) delivery For example, AXO-AAV-GM2 for Tay-Sachs disease or Sandhoff disease (https: / / clinicaltrials.gov / ct2 / show / NCT04669535) - Although improved, the constraints on vector distribution are similar to those of intrathecal delivery, and heterogeneous cortical distribution has been demonstrated. - ICM injection is considered a very rare technique today. - Known procedural risks include subarachnoid hemorrhage, brainstem injury, decreased vertebral artery blood flow, and death.

[0011] Subpiatric spinal cord delivery For example, AAV9-shRNA-SOD1 for ALS (Bravo-Hernandez et al., 2020). - The subpia space is the only possible space in the human spinal cord, and surgical access to it is difficult. - When targeting the subpia space, there is a high risk of nerve and vascular damage. - Spinal cord injuries carry a significant risk of permanent neurological damage, including paralysis. - CNS permeation is dependent on the concentration gradient and is uneven. - High risk of dorsal root ganglion toxicity. - Effective dosages for the motor cortex and subcortical motor nuclei have not been demonstrated.

[0012] Convection-facilitated delivery to brain parenchyma (CED) Intrathalamic delivery For example, intrathalamic CED of AAV2-GDNF (Kells et al., 2009). - The motor nuclei of the thalamus are the ventral anterior nucleus (VA) and the ventral lateral nucleus (VL). The VA nucleus receives afferent nerves from the basal ganglia and sends efferent nerves to the premotor cortex. The VL nucleus receives afferent nerves from the basal ganglia and dentate nucleus and sends efferent nerves to the premotor nucleus and primary motor nucleus. Because there are no direct motor axonal connections with the spinal cord or cranial nerve motor nuclei, the therapeutic benefit of targeting the thalamus alone for motor disorders may be limited. - The combined volume of the ventral anterior nucleus (VA) and ventral lateral nucleus (VL), which are motor nuclei of the thalamus, is approximately 1,400 mm³ per hemisphere. 3 Typically, when using the CED method, the distribution volume of the injector in the gray matter is approximately four times the injection volume, and therefore 1,400 mm³ for 350 μL. 3 This will cover the area. Because of the relatively small volume, the amount of vector that can be safely delivered is limited, reducing the likelihood of achieving therapeutic levels in the widely distributed musculoskeletal system. Damage to the thalamus is associated with an increased risk of conditions such as thalamic pain syndrome.

[0013] Combination of thalamic delivery and brainstem delivery: For example, CED of AAV2-hASM-HA for type A Niemann–Pick disease (Salegio et al., 2010). - In non-human primates, AAV2 delivered to the brainstem via the prefrontal trajectory in combination with delivery to the thalamus increases vector delivery to the cortex compared to delivery to only the thalamus. However, there was no report of transgene expression in the spinal cord and cerebellum.

[0014] Motor cortex delivery: For example, CED of optogenetic adeno-associated virus vectors to macaque cortex (Khateeb et al., 2019). Transgene expression was demonstrated in the cortex (and also in secondary motor neurons in an equivalent rat study, e.g., Nieuwenhuis et al., 2021), but this approach has several drawbacks. - It can only cover a small volume of the motor cortex. - The potential for clinical translation of this method is limited due to the convoluted and complex shape of the human motor cortex. - The thickness of the human motor cortex (only 1 - 4.5 mm) makes accurate targeting very difficult. - Transgene expression in the cerebellar circuit was not identified in such studies. - Similarly, white matter CED of AAV (Barua et al., 2013) resulted in transgene expression within the motor cortex but not in distal components of the motor pathway or cerebellar circuit.

[0015] [[ID=w28]] Importantly, none of these strategies can effectively target the cerebellar circuits that are fundamentally important for motor control, namely the dentate-ruble-thalamic circuit, the olivocerebellar circuit, or the cerebromorebellar circuit. Therefore, improved methods are still needed to deliver the required concentrations of drugs or other therapeutic agents to the CNS regions most needed for neurological conditions characterized by widespread neurodegeneration in structures required for effective motor-sensory integration.

[0016] In 2022, Salegio et al. reported that AAV5-GFP expression was observed in NHP after a single CED injection into the cerebellum via the posterior transtectonic orbit. GFP expression was identified in the cerebellum, DCN, and associated cerebellar tracts, but only inconsistent expression was identified in the spinal cord (1 out of 4 subjects). Notably, transgene expression in the motor cortex was not identified in any of the animals.

[0017] The connections between motor systems, their anatomical locations, and the fundamentally important influence of cerebellar circuits mean that the pons and middle cerebellar peduncle are uniquely positioned as target delivery sites for therapeutic drugs. Specifically, at the axial level of the middle cerebellar peduncle within the pons are the corticospinal tract, which connects the primary motor cortex to the thalamus and spinal cord; the corticobulbar tract, which connects the premotor cortex to the pontine nuclei; and the dentate nucleus, which connects to the cerebellum, motor thalamus, and inferior olivary nucleus. Furthermore, posterior to the corticospinal tract is the medial lemniscus, which contains secondary neurons of the posterior column (components of the sensory system that transmit proprioceptive sensation, vibrational sensation, fine touch, and two-point discrimination). Immediately nearby is the superior cerebellar peduncle, which connects to the dentate nucleus, and the thalamus, red nucleus, and pons are also the origins of the motor trigeminal nerve. [Overview of the project]

[0018] To address the shortcomings of the above-described drug delivery methods and routes of administration, the inventors investigated convective-enhanced delivery of viral vectors to the pons, targeting ascending, descending, and transverse white matter pathways, to leverage anterograde and retrograde transport and promote transgene expression in distal target structures from the transcerebellar middle cerebellar peduncle (MCP) orbital. Advantageously, this orbital can be used to deliver gene therapy drugs to the cerebellum and deep cerebellar nuclei (DCNs), in addition to delivery to the pons.

[0019] The inventors hypothesized that, due to the unique and extensive connectivity of nuclei and pathways in the pons and cerebellum, injection of viral vectors via the MCP, transported retrogradely and anterogradely into this relatively small CNS volume, has the potential to transduce all major neural circuits involved in sensorimotor processing. These include pyramidal and extrapyramidal motor pathways, as well as limbic, somatosensory, visual, and auditory circuits that modulate motor activity.

[0020] The inventors delivered viral vectors to the pons and cerebellum of sheep using a convection-enhanced delivery (CED) method via a cannula inserted in a trajectory from the cerebellum and MCP to the pons. Vectors AAV5 and AAV9, known to be transported axonally in both anterograde and retrograde directions, carried the transgene of the non-secreted fluorescent marker protein mCherry. One month after injection, postmortem analysis of the sheep's central and peripheral nervous systems demonstrated widespread transgene expression in the prefrontal cortex, parietal cortex, temporal cortex, and occipital cortex, thalamus and basal ganglia, hippocampus and amygdala, brainstem, spinal cord, cranial nerves including the optic tract, peripheral nerves including the sciatic nerve, and muscles. This novel route of administration of gene therapy agents to the CNS overcomes many of the limitations of alternative routes.

[0021] The broad CNS and peripheral nerve distribution covering major neural pathways for sensorimotor processing, which can be achieved by delivering anterograde and retrograde transported viral vectors to the pons and cerebellum via MCP orbitals, is due to the broadly distributed connectivity as follows:

[0022] Connectivity of the pons The pons is the intermediate part of the brainstem located between the midbrain (rostral) and the medulla oblongata (caudal). The pons contains cranial nerves and other nuclei, as well as the ascending, descending, and transverse pathways, with the following connectivity:

[0023] Cranial nerve nuclei: sensory nucleus of the trigeminal nerve, motor nucleus of the trigeminal nerve; abducens nerve nucleus; facial nerve nucleus; superior salivary nucleus; inferior salivary nucleus; vestibular nucleus; cochlear nucleus; nucleus tractus solitarius. The motor nuclei of the trigeminal nerve and facial nerve send efferent nerves to the masticatory muscles and facial expression muscles, respectively, and receive afferent nerves from the motor cortex to the corticomedulla oblongata. The vestibular nuclei receive inputs regarding position, movement, and balance from the semicircular canals via the vestibular branches of the vestibular-cochlear nerve, as well as from other regions including the spinal cord, the contralateral vestibular nuclei, and the reticular formation. These nuclei then relay the received information to regions of the brainstem, such as the reticular formation, oculomotor nuclei, thalamus, and cerebellum. The cochlear nucleus relays auditory information from the lateral lemniscus and inferior colliculus to the medial geniculate nucleus and auditory cortex. Efferent nerves from the inferior colliculus to the superior colliculus contribute to the tectospinal tract, which is part of the extrapyramidal system involved in orienting the eyes and head towards sound as part of auditory and visual reflexes. The nucleus tractus solitarius receives specialized sensory information related to taste, as well as general sensory input, from the tongue, palate, and pharynx via the facial nerve, glossopharyngeal nerve, and vagus nerve. Its efferent nerves are directed to the sensory thalamus and cortex, as well as to the brainstem reticular formation.

[0024] Other nuclei in the pons: pontine nuclei, lateral lemniscus nucleus, reticular nucleus of the pons, peduncleopontine nucleus, dorsolateral tegmental nucleus, locus coeruleus. The pontine nuclei receive extensive afferent nerves from the frontal, parieto-occipital, and temporal cortices, thereby projecting pontocerebellar fibers via the middle cerebellar peduncle to the contralateral cerebellar cortex and DCN, establishing the corticopontine-cerebellar pathway. The cerebellum plays a crucial role in cognitive and sensorimotor processing. Approximately 70-80% of the fibers in the middle cerebellar peduncle originate from the anterior frontal, temporal, parietal, and limbic lobes and are involved in cognition, while the remaining 20-30% originate from the sensorimotor cortex and are involved in motor processing. Therefore, retrograde transport of viral vectors along corticopontine fibers from pontine injection can provide a means to achieve extensive cortical transduction. The lateral lemniscus nucleus receives fibers from the cochlear nucleus and superior olivary nucleus complex and projects into the superior and inferior colliculi. The lateral lemniscus nucleus is involved in the coordination of auditory and visual responses. The pontine reticular formation (PRF) nuclei receive afferent nerves from the anterior frontal cortex, premotor cortex, motor cortex, parietal lobe, and temporal lobe. These cortical inputs provide information for motor planning and execution, sensory processing, and cognitive functions. Various brainstem nuclei, including the superior colliculus, raphe nuclei, locus coeruleus, and vestibular nuclei, send projections to the PRF. These connections are involved in the coordination of motor responses, the regulation of arousal and sleep-wake cycles, the modulation of pain perception, and the integration of sensory information. Interconnections between the intralaminar and ventromedial nuclei of the thalamus contribute to arousal regulation, attention, and sensory processing, while interconnections with the cerebellum contribute to the coordination of motor responses, the regulation of muscle tone, and the regulation of eye movements. The PRF receives input from the spinoreticular tract and corticoreticular tract. These pathways transmit sensory information from the body and limbs and contribute to motor control, reflex modulation, and pain processing. PRF sends efferent nerves to the anterior horn cells of the spinal cord, where it modulates muscle tone, reflex response, motor coordination, and the enhancement of antigravity muscle activity, while also serving as a pathway through which higher brain regions can influence motor activity at the spinal cord level. The peduncleopontine nuclei (PPN) receive afferent neurons from the cerebral cortex, including the primary motor cortex (M1), supplementary motor area (SMA), anterior frontal cortex, and sensory cortex. The basal ganglia include the globus pallidus, substantia nigra pars compacta (SNc), and subthalamic nucleus (STN). These connections are involved in regulating motor function, particularly in the context of motor initiation, coordination, and modulation. The PRF also receives afferent neurons from the cerebellar nuclei, as well as from the brainstem nuclei, including the reticular formation and vestibular nuclei. These connections contribute to arousal, attention, and modulation of sensory processing, as well as coordination of motor responses. The amygdala and hippocampus also send afferent neurons to the PPN, potentially influencing their role in emotional processing and motivational behavior. The PPN sends efferent neurons to the SNc, striatum, basal forebrain, amygdala, midbrain motor areas, pontine reticular formation, locus coeruleus, superior colliculus, and spinal motor pattern generators. The locus coeruleus projects fibers into various regions of the central nervous system, including the cerebral cortex, diencephalon, limbic system, brainstem, cerebellum, and spinal cord. The locus coeruleus plays a role in the wake-sleep cycle, attention, and stress response.

[0025] Transverse tract: Fibers from the cochlear nucleus and transverse pontine (pontocerebellar) fibers.

[0026] Ascending pathways: medial lemniscus, lateral lemniscus, spinal lemniscus (spinothalamic tract), trigeminal lemniscus (trigeminal nerve-thalamic tract). The medial lemniscus is formed by fibers from the gracile and cuneate nuclei within the dorsal medulla. The medial lemniscus transmits proprioceptive, vibratory, and fine touch sensations from the posterior column of the spinal cord to the VPL thalamus and ultimately to the primary somatosensory cortex. Retrograde transport of vectors from the gracile and cuneate nuclei is thought to be to nucleated primary sensory neurons in the dorsal root ganglia. The lateral lemniscus forms part of the auditory pathway, and its fibers terminate in the inferior colliculus. The lateral lemniscus receives afferent nerves from various nuclei, including the cochlear nucleus, the superior olivary nucleus, and the lateral lemniscus nucleus. The spinal lemniscus (spinothalamic tract) is formed from a combination of the anterior and lateral spinothalamic tracts. The spinal lemniscus transmits information about rough touch, pain, and temperature from the contralateral side of the body to the thalamus and primary somatosensory cortex. Retrograde transport is thought to occur to nucleated primary sensory neurons in the dorsal root ganglia of the spinal cord.

[0027] Descending tracts: corticospinal tract, corticobulbar tract, corticopontine tract, reticulospinal tract, rubrospinal tract, rubroreticular tract, vestibulospinal tract, and tectomedullary tract. The corticospinal tract primarily originates in the primary motor cortex, descends through the basal pons to the medulla, where most of the fibers cross to form the pyramidal decussation, and then continues contralaterally as the lateral corticospinal tract. The remaining fibers that do not cross remain ipsilateral and continue caudally as the anterior corticospinal tract. The fibers of the corticospinal tract synapse with anterior horn motor neurons in the spinal cord and are responsible for voluntary movement of the muscles of the trunk and limbs. The corticobulbar tract originates in the motor cortex of the brain and descends to the motor nuclei of cranial nerves in the pons and medulla. The corticobulbar tract is responsible for controlling voluntary movements of the face, head, and neck. The corticopontine tract is a large group of fibers that originate in the frontal cortex, parieto-occipital cortex, and temporal cortex, and terminate in neurons of the ipsilateral pontine nuclei, as described above. The pontine reticulospinal tract descends through the brainstem and spinal cord. It primarily terminates in the anterior horn of the spinal cord, where it forms synapses with lower motor neurons and interneurons. The pontine reticulospinal tract is particularly involved in facilitating and regulating spontaneous and reflexive movements related to posture and movement. The rubrospinal tract descends from the red nucleus, crosses over in the upper pons, and descends into the spinal cord, where it forms synapses with secondary motor neurons in the anterior horn. The rubrospinal tract is important for coordinating precise movements such as reaching for, grasping, and manipulating objects. The red-reticulous tract is mediated by the reticular formation and plays a role in modulating and coordinating motor activity that influences the activity of spinal reticulosomial neurons. The vestibulospinal tract descends to the spinal cord via lateral pathways, where it forms synapses with anterior horn cells, influencing the antigravity muscles of the limbs and trunk. The vestibulospinal tract has a medial component that primarily terminates in the neck and influences the cervical muscles, particularly those involved in head and neck orientation and stabilization. The tectomedullary spinal tract originates in the superior colliculus, receiving input from the retina and other visual pathways, and sending projections to the brainstem, particularly the reticular formation and cranial nuclei, as well as to the spinal cord. This pathway assists in the coordination of eye, head, neck, and body movements in response to visual stimuli, such as tracking moving objects or adjusting posture and maintaining balance in response to visual cues. Therefore, delivery of retrograde transport viral vectors that transduce the tectomedullary spinal tract and thus the superior colliculus can transduce the optic tract, retina, and visual cortex.

[0028] Compound tracts: medial longitudinal tract, central tegmental tract. The medial longitudinal fasciculus consists of both ascending and descending fibers that establish connections with the oculomotor nuclei, trochlear nuclei, abductor nucleus, Edinger-Westphal nucleus, vestibular nuclei, reticular nuclei, and spinal accessory nuclei. Through these connections and its connection with the superior colliculus, the medial longitudinal fasciculus plays a role in coordinating synovial eye movements and the associated head and neck movements. The central tegmental tract is a complex bundle of nerves that extends throughout the brainstem and terminates in the inferior olivary nucleus. The central tegmental tract includes descending fibers from the red nucleus that terminate in the ipsilateral inferior olivary nucleus, and ascending gustatory fibers from the nucleus tractus solitarius that terminate in the cortical gustatory region. Ascending and descending fibers of the brainstem reticular formation also pass through the central tegmental tract, and it serves as a pathway connecting the midbrain and cerebellum.

[0029] Connectivity of the middle cerebellar peduncle and cerebellum Injecting gene therapy vectors along the axis of the middle cerebellar peduncle (MCP) increases pontocerebellar pathway transmission from the contralateral pontine nuclei to the cerebellar cortex and deep cerebellar nuclei, and importantly, includes the inferior cerebellar peduncle and dentate nucleus. Due to the unique connectivity of the inferior cerebellar peduncle and dentate nucleus, their inclusion provides a means of transduction of several key structures essential for effective sensorimotor and cognitive processing, which is not thought to be achievable with injections limited to the pons.

[0030] The inferior cerebellar peduncle (ICP) serves as the primary pathway for transmitting sensory information from various sources to the cerebellum. The ICP contains several neural pathways, each with distinct connections and functions, including the following: 1. Spinocerebellar tract: • Dorsal spinocerebellar tract: This tract transmits unconscious proprioceptive sensory information from the lower limbs and trunk to the cerebellum, which originates in the Clark nucleus of the spinal cord and is essential for normal motor function. • Ventral spinocerebellar tract: Transmits proprioceptive sensory information from the lower limbs and trunk to the cerebellum. It originates from neurons in the spinal cord, crosses to the opposite side, and then ascends to the cerebellum. 2. Cuneate fasciculus cerebellar tract: • It transmits proprioceptive sensory information from the upper limbs and cervical region to the cerebellum. It originates in the Clark nucleus of the medulla oblongata. 3. Oliveocerebellar tract Climbing fibers: These originate from neurons in the inferior olivary nucleus of the medulla oblongata. Climbing fibers directly form synapses with Purkinje cells in the cerebellar cortex, providing strong excitatory input. These fibers are crucial for motor learning and coordination. 4. Vestibulocerebellar tract • Vestibular input is transmitted from the vestibular nuclei of the brainstem to the cerebellum. These fibers transmit information related to balance, spatial orientation, and head position.

[0031] The sensory information transmitted through these neural pathways is essential for the cerebellum's crucial functions of proprioception, balance, and coordination. Upon entering the cerebellum, fibers from these pathways synapse with neurons in the cerebellar cortex and deep cerebellar nuclei, contributing to the integration of sensory and motor signals necessary for motor control and coordination. The cerebellum then processes this information and transmits outputs to various motor centers in the brainstem and cerebral cortex, modulating motor commands and contributing to the fine-tuning of movement patterns.

[0032] The dentate nucleus receives most of its afferent nerves from the cerebellar cortex, but these are indirect. Other afferent nerves come from the contralateral pontine nuclei and inferior olivary nucleus, transmitting excitatory input and contributing to motor learning and coordination. Efferent nerves from the dentate gyrus pass through the superior cerebellar peduncle to the contralateral thalamus, particularly the ventrolateral thalamus (VL) and ventropretrolateral thalamus (VA), from where they proceed to the anterior frontal cortex, premotor cortex, and motor cortex. The dentate gyrus also has high functional connectivity (polysynaptic) to the amygdala (limbic system), as well as to the occipital, parietal, and frontal components, which are responsible for visual attention and spatial cognition. Other efferent nerves extend to the cerebellar cortex and the contralateral red nucleus and reticular formation. Dentate gyrus connections are involved in the integration of limbic system cognition and coordinated motor and sensory information, contributing to cognition, attentional control, visual and sensory processing, motor planning, sequencing, and coordination. Due to its extensive connectivity, the dentate nucleus is a highly effective target for distributing transgenes to larger areas of the cortex and subcortical structures using anterograde and retrograde transport vectors.

[0033] The pons is also larger in volume and generally oval-shaped (approximately 48,000 mm in adults). 3 ), which contributes to its usefulness as a target for gene therapy. This is, for example, the motor nucleus of the thalamus (1,400 mm 3 This is considerably larger than [another method]. It allows for the safe infusion of large volumes of therapeutic agents into the pons without causing harmful clinical effects (Szychot et al., 2021), thereby facilitating the optimization of the therapeutic dose range for gene therapy.

[0034] Accordingly, in a first aspect, the present invention provides a gene or RNA therapeutic vector for use in the treatment of non-malignant CNS disorders, which is to be injected into the pons by convection-enhanced delivery (CED). Also provided is a method for treating non-malignant CNS disorders, which comprises injecting the gene or RNA therapeutic vector into the pons by CED. Optionally, the gene or RNA therapeutic vector is also injected into the middle cerebellar peduncle.

[0035] Gene or RNA therapeutic vectors are administered by convection-enhanced delivery (CED). Convection-enhanced delivery is well known in the art. Convection-enhanced delivery generally refers to the delivery of a drug or other composition to the brain under a positive pressure gradient through a narrow catheter having an inner diameter of less than 500 μm, and more commonly less than 250 μm. Administration of gene or RNA therapeutic vectors or other compositions by CED typically results in a larger volume of distribution than that achieved by intracerebral injection or infusion.

[0036] Gene or RNA therapeutic vectors may be injected into the anterior pons. Preferably, the gene or RNA therapeutic vectors are injected into the pons and middle cerebellar peduncle. Preferably, the injection is performed from at least one cannula placed via a trans-cerebellar posterior to anterior trajectory that transversely crosses to the deep cerebellar nuclei. The inventors have demonstrated that by using this method, it is possible to achieve an injection fluid distribution encompassing the DCN without risking damage to these important structures, i.e., without the need to directly place the cannula into or through the DCN. In embodiments of the invention, the injection is performed via two bilaterally placed catheters, each catheter placed via a trans-cerebellar posterior to anterior trajectory that transversely crosses to the deep cerebellar nuclei. This trajectory can be used in conjunction with one or more transfrontal trajectories targeting the pons via an superior to inferior pathway.

[0037] The cannula may be a resistive cannula, such as a stepped cannula, as described, for example, in Gill et al., 2013 (incorporated herein by reference). Preferably, the injection is performed using a stepped cannula including a guide tube and a fluid transfer tube, the fluid transfer tube being inserted into the tissue through the guide tube. The guide tube creates a step where the fluid transfer tube emerges. More preferably, the fluid transfer tube includes four or more perfusion regions on its outer surface, configured to allow the injectable fluid to flow along the perfusion regions from the distal end of the fluid transfer tube toward the proximal end of the fluid transfer tube when the distal end of the fluid transfer tube is inserted into the tissue. The combination of stepped regions and perfusion regions can provide a more uniform and consistent distribution of the injectable fluid around the fluid transfer tube. Such a cannula is described in detail in British Patent No. 2307199.6, incorporated herein by reference.

[0038] A cannula step can be positioned at the interface between the posterior deep cerebellar nucleus (DCN) and the cerebellar white matter. In fact, the inventors have demonstrated that it is possible to control the perfusion of the injectable fluid by using a stepped cannula with a step at the posterior interface between the DCN and the cerebellar white matter. By perfusion of the injectable fluid back into the catheter step, it is possible to distribute the injectable fluid within the DCN without risking damage to these important structures, and in addition, to include the inferior cerebellar peduncle, which passes medial to the middle cerebellar peduncle at a more caudal lateral position. The inferior cerebellar peduncle transmits proprioceptive sensory information from the spinal cord, information from the inferior olivary nucleus related to motor timing and coordination, and vestibular nucleus transmission information related to balance and spatial orientation to the cerebellum.

[0039] Genetic or RNA therapeutic vectors may contain viral or non-viral vectors.

[0040] Suitable viral vectors include adenoviruses, adeno-associated viruses (AAVs), lentiviruses, and herpesviruses. Preferably, the viral vector is an AAV vector such as AAV5 or AAV9. Alternatively, the viral vector may be an AAV hybrid such as AAV2 / 5.

[0041] The most studied AAV serotypes in the CNS are serotypes 1, 2, 5, 6, 8, 9, and recombinant human (rh)10. A recombinant genome of a given serotype may be packaged in a capsid of another serotype (e.g., rAAV2 / 5 contains an AAV2 recombinant genome packaged in a capsid protein encoded by the AAV5 cap gene). AAV serotypes have been characterized in animal models and can be selected for targeted gene therapy based on specific tissue tropism, in vivo distribution, and transduction efficiency. Transfection of AAV serotypes 2 and 6 is limited to neurons, while serotypes 7, 9, and 5 transfect both neurons and astrocytes. Axonal transport of AAV transgene capsids is either anterograde or retrograde, varies by serotype, and may be target and dose-dependent. AAV6 typically exhibits retrograde axonal transport, while AAV1, 2, 5, 8, and 9 have been shown to have either anterograde or retrograde axonal transport, depending on the transfected neuron type and / or the dose of vector delivered to the CNS tissue (Li et al., 2019).

[0042] When gene or RNA therapeutics are unilaterally injected into the pons and middle cerebellar peduncle using virus vectors that are transported both anterogradely and retrogradely, due to the unique connectivity of the structures involved in the injection, they can be distributed bilaterally in the cerebral cortex and cerebellar cortex. This is because unilateral injection in the pons that transduces the ipsilateral pontine nucleus will distribute retrogradely to the widely spreading corticocentric nerves and anterogradely to the contralateral cerebellar cortex and DCN. The pontocerebellar fibers passing from the contralateral side along the ipsilateral middle cerebellar peduncle are also transduced, resulting in anterograde transport to the ipsilateral cerebellum and DCN and retrograde transport to the contralateral cortex along the corticocentric nerves. Anterograde transport along the centrifugal nerve fibers from the cerebellar cortex and DCN has significant connectivity with the cerebral cortex and subcortical structures, thereby significantly enhancing the distribution of the therapeutic transgene or RNA therapeutics. The pons provides a unique anatomical location where the pyramidal and extrapyramidal nerve pathways converge, facilitating transduction in a relatively small CNS volume and anterograde and retrograde transport to the cortex, subcortical structures, and spinal cord. Similarly, the ascending somatosensory pathway can be transduced in the pons and distributed to the cortex and spinal cord, except for the dorsal spinocerebellar tract that passes through the inferior cerebellar peduncle to reach the cerebellum. However, as described above, the inferior cerebellar peduncle can be covered by injection along the middle cerebellar peduncle. Finally, the unique connectivity of the pons provides a location where transduction of special sensory pathways can also be achieved, as described above. If necessary, bilateral injection can further enhance CNS distribution. This makes it possible to administer an effective amount of the vector widely distributed in the CNS structures while reducing the concentration of the vector delivered from a single cannula, thereby reducing the potential for local toxicity.

[0043] The gene or RNA therapy vector is about 1×10 per ml 3 virus particles ~ about 1×10 per ml 20 virus particles, or about 1×10 per ml 5 virus particles ~ about 1×10 per ml 15 virus particles, preferably about 1×10 per ml 5Virus particles: approximately 1 x 10 per 1 ml 15 Virus particles, or approximately 1 x 10⁶ per 1 ml 8 ~Approx. 1×10 13 The virus particles can be administered at a concentration. The CED flow rate will typically be in the range of 0.5 μL / min to 20 μL / min or 1 μL / min to 20 μL / min, preferably 1 μL / min to 10 μL / min, and more preferably 1 μL / min to 5 μL / min. In embodiments of the present invention, the flow rate may be about 3 μL / min.

[0044] The injection volume may be about 50 μl to about 10 mL, or about 0.5 mL to about 5 mL, preferably about 0.5 mL to about 3 mL, more preferably about 0.5 mL to about 2 mL. In embodiments of the present invention, the injection volume may be about 1 mL, or about 0.5 mL, or about 0.3 mL. The injection volumes described herein are usually daily volumes.

[0045] The injection volume from a single cannula may be approximately 0.5 mL to 3 mL. In this strategy, one cannula can be inserted into each middle cerebellar peduncle, so the total injection volume may be up to 6 mL. When two additional cannulas are inserted into the cerebral peduncle, these cannulas are preferably directed toward the median sagittal portion of the pons, with one cannula positioned more anteriorly to deliver to the base of the pons and the other more posteriorly to deliver to the tegmentum of the pons. With this latter positioning, four cannulas can be injected simultaneously, and the total injection volume to the pons, MCP, and cerebellum is up to 10 mL. The injection is preferably performed in an awake patient so that the patient can be neurologically monitored. The distribution of the injectable fluid during injection can also be monitored with intermittent MRI scans. Optionally, gene therapy vectors are co-injected with MRI contrast agents such as gadolinium-based contrast agents (GBCAs) including gadopentate dimeglumine (MultiHance), gadobutrol (Gadavist), gadopentetate dimeglumine (Magnevist), gadoteridol (ProHance), and gadoterate meglumine (Dotarem). Alternative MRI contrast agents include iron oxide nanoparticles, ultra-small superparamagnetic iron oxide particles, or manganese-based contrast agents.

[0046] Suitable non-viral vectors include polymers, lipids, peptides, inorganic materials, and hybrid systems. Polymer vectors may be non-biodegradable (e.g., polyethyleneimine (PEI), poly(vinylimidazole) (PVI), and PAMAM) or biodegradable (e.g., chitosan, poly(β-aminoester) (PBAE), and polylactic acid (PLA)). Examples of lipid vectors include conventional lipids, gemini-type surfactants, lipidoids, and helper lipids. Peptide vectors include peptide nucleic acid conjugates, polypeptides, and cell-penetrating peptides. Inorganic vectors include silica-based systems such as mesoporous silica nanoparticles, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, graphene, upconversion nanoparticles, and quantum dots. Examples of hybrid vector systems include inorganic-organic hybrid vectors, modified PEI vectors, inorganic-lipid vectors, and peptide-lipid vectors.

[0047] Gene or RNA therapy, as is well known, involves using genetic material to modulate or add genes to an individual's cells to treat a disease. The genetic material introduced may be any suitable genetic material, including DNA, RNA, small interfering RNA (siRNA), microRNA, short hairpin RNA (shRNA), or antisense oligonucleotide (AON) cassettes. In embodiments of the present invention, the gene or RNA therapy may include CRISPR / Cas9. Using gene or RNA therapy, diseases can be treated in any known way, including gene substitution, gene knockdown, gene editing, pro-survivability gene therapy, and cell suicide therapy, as well as the expression of neuroprotective and / or nerve repair molecules such as neurotrophic factors. In embodiments of the present invention, the gene or RNA therapy agent may be a nucleotide sequence encoding a monoclonal antibody capable of expressing an antibody-protein fusion product such as full-length IgG, immunoadhesin, or bispecific substance, or an antibody fragment such as an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), or a single-domain antibody.

[0048] A combination of gene and / or RNA therapeutic vectors may be administered. For example, two different vectors, each containing the same or different genetic material, may be administered sequentially or in combination (i.e., simultaneously). If the first vector induces or risks inducing an immune response, it may be advantageous to administer the two different vectors sequentially. By using different vectors to deliver further genetic material, the induction of an immune response can be avoided. In addition or alternatively, two or more different gene or RNA therapeutic agents may be administered using the same vector. Two or more gene or RNA therapeutic agents may be delivered sequentially or in combination (i.e., simultaneously).

[0049] The gene and / or RNA therapeutic vectors described herein may be administered in combination with one or more additional gene or RNA therapeutic vectors described above, and / or in combination with one or more other therapeutic agents such as histone deacetylase inhibitors or albumin. The additional gene and / or RNA therapeutic vectors or other therapeutic agents may be administered systemically, intracerebrospinal fluid (CSF), intraparenchymal, or subpia.

[0050] The present invention can be used to treat neurological disorders characterized by pathological changes in motor (pyramidal and extrapyramidal) pathways, cerebellar pathways, sensory and limbic pathways, and the cerebral cortex. Therefore, therapeutic indications are considered according to these target structures, but in most of the disorders listed, the pathological changes are not limited to a single component of the nervous system, but in fact affect components of the cortical and subcortical motor, sensory, and coordination networks, requiring broad distribution of the therapeutic agent.

[0051] A disease characterized primarily by degeneration of the pyramidal motor pathway. Amyotrophic lateral sclerosis (ALS), formerly known as Lou Gehrig's disease, can affect upper and lower motor neurons. This disease leads to a rapid loss of muscle control and ultimately paralysis. While motor neuron degeneration is a prominent feature of ALS, loss of sensory pathways also affects patients and may be associated with cognitive impairment.

[0052] Spinal muscular atrophy (SMA) refers to a group of genetic disorders that affect lower motor neurons. The most common form is caused by a mutation or deletion in a gene known as the survival motor neuron gene 1 (SMN1), which leads to neuronal degeneration, resulting in muscle weakness and wasting.

[0053] Spinal muscular atrophy type 1 with respiratory distress (SMARD1) is a very rare form of spinal muscular atrophy (SMA) caused by a mutation in the IGHMBP2 (immunoglobulin helicase μ-binding protein 2) gene. Symptoms appear in infancy, between 6 weeks and 6 months of age. Children with SMARD1 may experience sudden respiratory failure due to diaphragmatic paralysis and may develop muscle weakness in their limbs.

[0054] Congenital SMA with joint contractures is a rare disorder that manifests at birth. Symptoms include severe joint contractures, which prevent infants from extending or flexing the affected joints. In most children, both the arms and legs are affected. Other symptoms include scoliosis (curvature of the spine), thoracic deformities, respiratory problems, an abnormally small jaw, and drooping eyelids.

[0055] Progressive bulbar palsy (PBP), also known as progressive bulbar atrophy, is caused by damage to the upper motor neurons in the brainstem or the lower motor neurons connected to the brainstem. The brainstem controls the muscles necessary for swallowing, speaking, chewing, and other functions.

[0056] Primary lateral sclerosis (PLS) affects only the upper motor neurons, causing difficulty and slowness of movement in the arms, legs, and face. Symptoms include weakness, muscle rigidity, and spasticity, clumsy movements, slowness of movement, and problems with balance and speech. The disorder often affects the legs first, followed by the torso, arms, and hands, and ultimately the muscles used for swallowing, speaking, and chewing.

[0057] Progressive muscular atrophy (PMA) is a rare subtype of ALS characterized by slow but progressive lower motor neuron damage. It is more common in men than women and usually begins at a later age than typical ALS. People with PMA typically notice weakness in their hands or feet, followed by weakness in other areas of the body. They may also experience weakness in the torso muscles and difficulty breathing. Exposure to cold can worsen an individual's symptoms. Other symptoms may include muscle wasting or atrophy, clumsy hand movements, spasms, and muscle cramps.

[0058] Kennedy disease is a hereditary lower motor neuron disorder that affects men. The onset of symptoms varies, but it usually begins between the ages of 20 and 40. Kennedy disease is also known as spinal muscular atrophy (SBMA), bulbar spinal muscular atrophy, or X-linked spinal muscular atrophy. The disease is caused by a mutation in the androgen receptor gene. Daughters of people with Kennedy disease have a 50% chance of having a son with the disease.

[0059] Post-polio syndrome (PPS) typically develops 15 to 40 years after an individual has had polio, an infectious viral disease. PPS is thought to be the result of long-term degeneration of motor neurons, leading to muscle weakness and functional impairment. PPS is not contagious, and only those who have had polio may develop it; not everyone who has had polio will develop PPS.

[0060] A disease characterized primarily by degeneration of the extrapyramidal motor pathway. Parkinson's disease and Parkinson's disease plus syndrome are characterized by muscle rigidity, resting tremor, and slowness of movement. In addition, autonomic dysfunction and cognitive impairment are also common. Extensive, progressive pathological changes occur, starting in the lower brainstem and ascending to the midbrain, amygdala, thalamus, and finally the cerebral cortex.

[0061] Huntington's disease is a genetic disorder that affects behavior, cognition, and movement, causing uncontrolled, rapid, convulsive limb movements. Huntington's disease is caused by a defect consisting of an elongation of the CAG repeat in the huntingtin gene (HTT).

[0062] Dystonia is a hyperactive movement disorder characterized by involuntary movements and slowness of intended movements. While metabolic, vascular, and structural abnormalities are known causes of dystonia, there are still many cases where the cause is unknown. Dystonia can occur as a hyperactive disorder or as a side effect of hypokinetic disorders such as Parkinson's disease.

[0063] Hemiballism is a hyperactive movement disorder that causes uncontrolled movements on one side of the body. Hemiballism is generally caused by damage to the subthalamic nucleus (STN).

[0064] Tourette syndrome is a disorder characterized by behavioral and motor tics, OCD, and attention deficit hyperactivity disorder. Tourette syndrome is thought to be caused by abnormalities in the brain's motor network, limbic network, and collaborative network.

[0065] Sydenham's chorea is a disorder characterized by rapid, uncoordinated spasms, primarily affecting the face, hands, and feet. Sydenham's chorea is the result of an autoimmune response following infection with Group A beta-hemolytic streptococci (GABHS), leading to damage to the basal ganglia.

[0066] Motor cerebral palsy is a subtype of cerebral palsy associated with damage to the basal ganglia during brain development due to bilirubin encephalopathy and hypoxic-ischemic brain injury. Symptoms include slow, uncontrolled movements of the limbs and trunk, as well as small, rapid, random, repetitive, uncontrolled movements known as chorea. Involuntary movements often increase during periods of emotional stress or agitation and disappear when the patient is asleep or distracted.

[0067] Athymormic syndrome is a rare psychopathological and neurological syndrome characterized by extreme passivity, apathy, emotional blunting, and a severe general loss of self-motivation. It is thought to be caused by damage to the basal ganglia or prefrontal cortex, particularly the striatum and globus pallidus, which are responsible for motivation and executive function.

[0068] Lesch-Nyhan syndrome is a rare X-linked recessive disorder caused by a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), resulting in uric acid accumulation and dopamine deficiency. Within the first few years of life, extrapyramidal complications occur, causing abnormal involuntary muscle contractions such as loss of motor control (dystonia), distressing movements (chorea athetosis), and arching of the spine (opisthotonus).

[0069] Wilson's disease is an autosomal recessive genetic disorder caused by a mutation in the copper transport gene ATP7B, leading to excessive copper accumulation. Neurological symptoms include Parkinsonian symptoms (most commonly cogwheel rigidity, bradykinesia or slowness of movement, or loss of balance), which may or may not be accompanied by typical hand tremors, mask-like facial expressions, slurred speech, ataxia, or dystonia.

[0070] A disease characterized primarily by degeneration of the cerebral cortex. Alzheimer's disease is the most common form of dementia and is caused by the degeneration of areas of the cerebral cortex that control memory, language, reason, and social behavior.

[0071] Frontotemporal dementia is primarily characterized by neurodegeneration in the frontal and temporal lobes of the brain.

[0072] Creutzfeldt-Jakob disease is a rare degenerative brain disorder that causes rapidly progressing dementia.

[0073] A disease characterized by marked degeneration of the cerebellar system. Friedreich's ataxia (FA) is an autosomal recessive genetic disorder with endocrine, cardiac, and nervous system manifestations, caused by mutations in the FXN gene on chromosome 9. The FXN gene produces frataxin, a protein involved in iron-sulfur protein synthesis as part of normal mitochondrial homeostasis. In most cases, FA is caused by a GAA trinucleotide repeat disorder in both alleles of the gene, leading to transcriptional defects in the FXN gene and thus reduced frataxin levels. Gene therapy targeting increased frataxin levels holds great potential for therapeutic benefit.

[0074] FA is the most common hereditary ataxia (affecting coordination, balance, and speech). Neurological symptoms typically appear by age 20 and include difficulty walking, proprioceptive and sensory abnormalities, weakness and muscle atrophy, and spasticity. Loss of the spinal dorsal root ganglia and degeneration of the peripheral nerves and spinal posterior column (sensory pathways) impairs the cerebellum's ability to coordinate movements. The dorsal spinocerebellar tract, dentate nucleus, cortical motor neurons, and corticospinal (motor) tract also degenerate. In FA, Purkinje cells in the cerebellum are also damaged. Purkinje cells are the sole output neurons of the cerebellar cortex, and their degeneration has serious adverse effects on cerebellar function. Some individuals may also experience visual impairment due to retinal degeneration.

[0075] Spinocerebellar ataxia is characterized by cerebellar atrophy causing ataxia, loss of motor neurons resulting in motor impairment, atrophy of the pons and medulla, and atrophy of the basal ganglia, and is therefore another potential therapeutic target for pontine delivery.

[0076] Diseases characterized by limbic system disorders Epilepsy is a brain disorder characterized by recurrent seizures of unknown cause.

[0077] Limbic encephalitis is a paraneoplastic phenomenon resulting from antibody-mediated limbic system inflammation. Its clinically prominent feature is the development of significant memory impairment (amnesia), often accompanied by irritability and behavioral disturbances, over a period of several days to several weeks.

[0078] Dementia is a group of diseases characterized by a decline in cognitive function that impairs daily living activities.

[0079] Anxiety disorders are a group of mental health conditions that include generalized anxiety disorder, panic disorder with or without agoraphobia, specific phobias, agoraphobia, social anxiety disorder, separation anxiety disorder, and selective mutism.

[0080] Schizophrenia is a serious mental disorder in which people interpret reality abnormally. Schizophrenia can be characterized by hallucinations, delusions, and extreme thought and behavioral disorders that impair daily functioning and can cause physical impairment.

[0081] Autism spectrum disorder (ASD) is a developmental disorder caused by differences in the brain. People with ASD often have difficulty communicating and interacting with others, and their behaviors or interests are restricted or repetitive. People with ASD may also have differences in how they learn, perform actions, or pay attention.

[0082] A disease characterized by widespread pathological changes throughout the central nervous system (CNS). Neurodegenerative diseases not listed elsewhere Lewy body disease, multiple system atrophy, corticobasal degeneration, hypertrophic olivary degeneration, and leukodystrophy including Alexander disease, autosomal dominant leukodystrophy with autonomic nervous system disorders, Canavan disease, cerebral tendon xanthomatous disease, childhood ataxia with central nervous system myelin dysplasia, Krabbe disease, metachromatic leukodystrophy, Pelizaeus-Merzbach disease, Refsum's disease (adult or infant), Rett syndrome and progressive supranuclear palsy, myelin dysplasia with basal ganglia and cerebellar atrophy, and essential tremor.

[0083] Multiple system atrophy is characterized by degeneration of the basal ganglia, inferior olivary nucleus, and cerebellum, resulting in autonomic dysfunction, tremors, bradykinesia, rigidity, postural instability, and ataxia.

[0084] Pelizaeus-Merzbach disease is an X-linked recessive leukodystrophy caused by mutations in proteolipid genes. This results in abnormalities throughout the white matter of the brain and spinal cord.

[0085] Myelin dysplasia, with atrophy of the basal ganglia and cerebellum, is a rare genetic disorder that causes progressive degeneration of the basal ganglia and cerebellum. It is a type of white matter dystrophy caused by mutations in the TUBB4A gene.

[0086] Enzyme deficiency Lysosomal storage disorders, including Teysachs disease, Sandhoff disease, neurogenic ceroid lipofuscinosis including Batten disease, Hunter syndrome, Haller disease, Niemann-Pick disease, neuronal axonal dystrophy, Gaucher disease, and Sanfilippo syndrome (mucopolysaccharidosis type III).

[0087] Neuroinflammatory diseases include multiple sclerosis, neurosarcoidosis, encephalitis, prion diseases, and neuromyelitis optica (Devic's disease).

[0088] In addition to those listed elsewhere, other neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD), affective disorders, Asperger's syndrome, and addiction.

[0089] Acquired neurological disorders, including stroke, traumatic brain injury, and spinal cord injury.

[0090] Potential gene therapies for neurological diseases, for example, delivered via the transmiddle cerebellar peduncle orbit to the pons and DCN by CED. Potential gene therapies for amyotrophic lateral sclerosis (ALS): Several gene therapy approaches for ALS are being studied, targeting different aspects of the disease. Some potential gene therapy strategies for ALS include the following: 1. GDNF (Glial Cell Line-Derived Neurotrophic Factor): GDNF is a protein that supports the survival of nerve cells. Gene therapy involving GDNF delivery aims to prevent motor neuron degeneration in ALS. 2. IGF-1 (Insulin-like Growth Factor 1): IGF-1 has neuroprotective effects and can help promote motor neuron survival. Gene therapy delivering IGF-1 aims to support motor neuron survival in ALS. 3. SOD1 (Superoxide Dismutase 1): Mutations in the SOD1 gene are associated with some cases of familial ALS. Gene therapy targeting SOD1 aims to reduce the toxic effects of mutant SOD1 protein on motor neurons. 4. ALS2 (Alsin Rho Guanine Nucleotide Exchange Factor ALS2): Mutations in the ALS2 gene are associated with early-onset ALS. Gene therapy targeting ALS2 aims to address the underlying genetic cause of ALS. 5. C9orf72 (Open Reading Frame 72 on Chromosome 9): Elongation of the C9orf72 gene is the most common genetic cause of familial ALS. Gene therapy targeting C9orf72 aims to reduce the toxic effects of the elongated repetitive sequence.

[0091] Potential gene therapy for Friederich's ataxia (FA): Friedreich's ataxia (FA) is a genetic disorder caused by mutations in the FXN (frataxin) gene. Frataxin is involved in mitochondrial function, and its deficiency leads to mitochondrial dysfunction and oxidative stress, particularly affecting the nervous system and heart.

[0092] Potential gene therapy approaches for Friedreich's ataxia aim to address the underlying frataxin deficiency. Some strategies being explored include: 1. Frataxin gene substitution: Gene therapy techniques may include delivering a functional copy of the FXN gene to cells affected by Friedreich's ataxia. This may potentially restore frataxin expression and alleviate the symptoms of the disease. 2. Frataxin gene enhancement: Another approach is to enhance the expression of residual functional FXN genes within cells. This may include increasing frataxin production through techniques such as gene editing or the use of viral vectors. 3. Small molecule therapy: While not strictly gene therapy, small molecule compounds are being studied to upregulate frataxin expression or enhance mitochondrial function in Friedreich's ataxia. 4. Antioxidant therapy: Considering the role of oxidative stress in the pathology of Friedreich's ataxia, antioxidant therapy is also being explored as a potential treatment.

[0093] Potential gene therapy for spinocerebellar ataxia type 1 (SCA1) Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder caused by the elongation of the CAG repeat in the ATXN1 gene, leading to the production of mutant ataxin 1 protein. This mutant protein disrupts normal cellular function, particularly affecting the cerebellum and causing progressive neuronal degeneration.

[0094] Gene therapy for SCA1 is still in the preclinical and early clinical stages, but several potential transgenes and techniques are being explored. 1. Silencing mutant ATXN1: One method is to selectively silence the expression of the mutant ATXN1 gene using RNA interference (RNAi) or antisense oligonucleotides (ASOs). This involves delivering small RNA molecules that target and degrade mutant RNA, thereby reducing the production of toxic mutant attaxin 1 protein. 2. Gene Editing: CRISPR-Cas9 and other gene editing technologies hold promise for correcting the underlying gene mutation in SCA1. Using gene editing tools, the DNA sequence of the ATXN1 gene can be edited more precisely by either removing extended repetitive sequences or replacing them with the correct sequence. 3. Transgene delivery: Another approach involves delivering a normal copy of the ATXN1 gene to affected cells. This can be achieved by using a viral vector, such as adeno-associated virus (AAV), to deliver the transgene to the cerebellum and other affected brain regions. The normal ATXN1 gene would then produce functional attaxin 1 protein, potentially alleviating the symptoms of SCA1. 4. Neuroprotective Factors: In addition to targeting the mutant ATXN1 gene, gene therapy strategies for SCA1 may also include delivering neuroprotective factors or molecules that promote cell survival and function in the cerebellum and other affected areas of the brain.

[0095] Potential gene therapies for neuronal ceroid lipofuscinosis 5 (CLN-5): CLN5, or neuronal ceroid lipofuscinosis 5, is a form of neuronal ceroid lipofuscinosis, a group of rare hereditary neurodegenerative disorders characterized by the accumulation of fatty pigments in cells, particularly neurons. Currently, there are no approved gene therapy treatments for CLN5, but research is underway to develop potential therapies.

[0096] Since CLN5 is a genetic disorder caused by mutations in the CLN5 gene, potential gene therapy approaches will likely involve addressing the underlying genetic defect. Some strategies that may be explored include: 1. Gene replacement therapy: This involves delivering a functional copy of the CLN5 gene to affected cells. This can be achieved by using a viral vector, such as adeno-associated virus (AAV), to deliver a normal CLN5 gene to neurons in the brain. Upon delivery, the normal CLN5 gene will produce a functional CLN5 protein, potentially correcting the underlying genetic defect and reducing the accumulation of fatty pigments. 2. Gene Editing: Gene mutations in the CLN5 gene can be corrected using CRISPR-Cas9 and other gene editing technologies. By using gene editing tools, the DNA sequence of the CLN5 gene in affected cells can be precisely edited, either by correcting the mutation or by replacing the defective gene with a normal copy. 3. Enhancement of Autophagy and Lysosomal Function: Since CLN5 is involved in lysosomal function, another potential gene therapy approach is to enhance autophagy and lysosomal function in affected cells. This may involve delivering genes encoding lysosomal enzymes or proteins that promote autophagy to neurons in the brain, thereby helping to remove accumulated fatty pigments and alleviate symptoms. 4. Neuroprotective Factors: Gene therapy strategies for CLN5 may also include delivering neuroprotective factors or molecules that promote brain cell survival and cellular function. This can mitigate neuronal loss and help slow disease progression.

[0097] Potential gene therapies for Niemann-Pick C1 type: Niemann-Pick disease type C1 (NPC1) is a rare lysosomal storage disorder caused by mutations in the NPC1 gene, which leads to impaired cholesterol transport within cells. Currently, there are no approved gene therapies for NPC1, but several approaches are being explored. 1. NPC1 gene replacement therapy: Gene therapy may involve delivering a functional copy of the NPC1 gene to affected cells. This approach aims to restore normal NPC1 protein function and thereby correct the underlying genetic defect. Viral vectors such as adeno-associated virus (AAV) or lentivirus can be used to deliver the normal NPC1 gene to cells in the brain and other affected tissues. 2. Gene Editing: CRISPR-Cas9 and other gene editing technologies offer the possibility of correcting gene mutations in the NPC1 gene. Using gene editing tools, the DNA sequence of the NPC1 gene in affected cells can be precisely edited, either by correcting mutations or by replacing defective genes with normal copies. 3. Cholesterol Metabolism Modulation: Since NPC1 affects intracellular cholesterol transport, gene therapy techniques may aim to modulate cholesterol metabolism to reduce its accumulation in lysosomes. This may include delivering genes encoding enzymes or proteins involved in cholesterol metabolism to affected cells to help restore normal cholesterol levels and alleviate symptoms. 4. Enhancement of Lysosomal Function: Since NPC1 primarily affects lysosomal function, gene therapy strategies may include enhancing the lysosomal function of affected cells. This can be achieved by delivering genes encoding lysosomal enzymes or proteins that promote lysosome biosynthesis and function, thereby aiding in the removal of accumulated lipids and alleviation of symptoms. 5. Neuroprotective Factors: Gene therapy techniques for NPC1 may also include delivering neuroprotective factors or molecules that promote brain cell survival and cellular function. This can help mitigate neuronal cell loss and slow the progression of neurological symptoms associated with the disease.

[0098] Potential gene therapies for multiple system atrophy (MSA): Multiple system atrophy (MSA) is a rare neurodegenerative disorder characterized by the progressive degeneration of specific nerve cells in the brain, leading to symptoms such as autonomic dysfunction, motor impairment, and coordination problems. Currently, there are no approved gene therapy treatments for MSA. However, research into potential gene therapy approaches is ongoing. Some potential strategies are listed below. 1. Neurotrophic Factors: Gene therapy may involve delivering genes encoding neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF). These factors may promote neuronal survival and function and slow the progression of neurodegeneration in MSA. 2. Alpha-synuclein regulation: MSA is characterized by the accumulation of alpha-synuclein aggregates in the brain. Gene therapy techniques may aim to reduce its accumulation and toxicity by regulating alpha-synuclein expression or clearance. This may include delivering genes encoding proteins involved in the alpha-synuclein degradation pathway, or using RNA interference (RNAi) to silence alpha-synuclein expression. 3. Enhancing Mitochondrial Function: Dysfunction of mitochondria, the cellular energy-producing organelles, has been suggested to be involved in the pathogenesis of MSA. Gene therapy strategies may include enhancing mitochondrial function by delivering genes encoding mitochondrial proteins or factors that promote mitochondrial biosynthesis and function. 4. Enhancement of Lysosomal Function: MSA is also associated with lysosomal dysfunction, leading to the accumulation of toxic substances within cells. Gene therapy techniques may aim to enhance lysosomal function by delivering genes encoding lysosomal enzymes or proteins that promote lysosomal biosynthesis and function. 5. Neuroinflammation Modulation: Brain inflammation is thought to contribute to neurodegeneration in MSA. Gene therapy strategies may include modulating neuroinflammatory pathways by delivering genes encoding anti-inflammatory factors or factors that promote the resolution of inflammation.

[0099] Potential gene therapies for progressive supranuclear palsy (PSP): Progressive supranuclear palsy (PSP) is a rare neurodegenerative disorder characterized by the accumulation of abnormal tau protein in specific areas of the brain, leading to motor and cognitive impairments. Currently, there are no approved gene therapy treatments for PSP. However, research is ongoing, and several potential gene therapy approaches are being explored. Some possibilities are listed below: 1. Tau Protein Modulation: PSP is characterized by the accumulation of abnormal tau protein in neurons. Gene therapy techniques may aim to reduce its accumulation and toxicity by modulating tau protein expression or clearance. This may include delivering genes encoding proteins involved in the tau degradation pathway or silencing tau expression using RNA interference (RNAi). 2. Neurotrophic Factors: Gene therapy may involve delivering genes encoding neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF). These factors may promote neuronal survival and function and slow the progression of neurodegeneration in PSP. 3. Enhancement of Lysosomal Function: Lysosomal dysfunction is suggested to be involved in the pathogenesis of PSP and is linked to the accumulation of toxic substances in cells. Gene therapy methods may aim to enhance lysosomal function by delivering genes encoding lysosomal enzymes or proteins that promote lysosomal biosynthesis and function. 4. Enhancing Mitochondrial Function: Dysfunction of mitochondria, the cellular energy-producing organelles, has been suggested to be involved in PSP. Gene therapy strategies may include enhancing mitochondrial function by delivering genes encoding mitochondrial proteins or factors that promote mitochondrial biosynthesis and function. 5. Neuroinflammation Modulation: Brain inflammation is thought to contribute to neurodegeneration in PSP. Gene therapy strategies may include modulating neuroinflammatory pathways by delivering genes encoding anti-inflammatory factors or factors that promote the resolution of inflammation.

[0100] Potential gene therapies for Rett syndrome: Rett syndrome is a rare genetic neurological disorder caused by mutations in the MECP2 gene, which plays a crucial role in the development and function of the nervous system. Currently, there are no approved gene therapy treatments for Rett syndrome, but research is ongoing, and several potential gene therapy approaches are being explored. Some of these possibilities are listed below: 1. MECP2 Gene Replacement Therapy: Rett syndrome is primarily caused by mutations in the MECP2 gene, leading to a reduction or loss of functional MECP2 protein levels. Gene therapy techniques may involve delivering a functional copy of the MECP2 gene to affected cells, aiming to restore normal MECP2 protein levels and function. Viral vectors, such as adeno-associated virus (AAV), are commonly used to deliver the gene to brain cells. 2. MECP2 Gene Editing: Gene editing technologies such as CRISPR-Cas9 offer the possibility of directly correcting gene mutations in the MECP2 gene. Using gene editing tools, the DNA sequence of the MECP2 gene in affected cells can be precisely edited, either by correcting mutations or by replacing defective genes with normal copies. 3. MECP2 Protein Modulation: Gene therapy techniques may also aim to modulate MECP2 protein expression or function. This may involve delivering genes encoding factors that regulate MECP2 expression levels or enhance the stability and activity of the MECP2 protein. 4. Neurotrophic Factors: Rett syndrome is associated with abnormalities in brain development and function. Gene therapy may involve delivering genes encoding neurotrophic factors such as brain-derived neurotrophic factor (BDNF) or insulin-like growth factor 1 (IGF-1), which promote neuronal survival, growth, and function. 5. Synaptic Enhancement: Rett syndrome is characterized by abnormalities in synaptic function, which is the exchange of information between neurons. Gene therapy strategies may aim to enhance synaptic function by delivering genes that encode proteins involved in synaptic transmission and plasticity.

[0101] Potential gene therapies for Sanfilipo syndrome: Sanfilippo syndrome, also known as mucopolysaccharidosis type III (MPSIII), is a group of rare genetic disorders caused by mutations in genes encoding enzymes involved in the breakdown of glycosaminoglycans (GAGs). There are four subtypes of Sanfilippo syndrome (A, B, C, and D), each caused by a different gene mutation.

[0102] Currently, there are no approved gene therapy treatments for Sanfilippo syndrome. However, research is ongoing, and several potential gene therapy approaches are being explored. Some possibilities are listed below: 1. Enzyme Replacement Therapy (ERT): Enzyme replacement therapy involves delivering a functional copy of the deficient enzyme to affected cells or tissues. In the case of Sanfilippo syndrome, this involves delivering the enzyme that is deficient in the specific subtype of the disease (e.g., heparan N-sulfatase in MPS IIIA, alpha-N-acetylglucosaminidase in MPS IIIB). ERT is already used to treat some other types of mucopolysaccharidosis (MPS). 2. Gene Replacement Therapy: Gene therapy techniques may involve delivering a functional copy of a mutant gene to affected cells. This can be achieved by using a viral vector, such as adeno-associated virus (AAV), to deliver a normal gene to cells in the brain and other affected tissues. For example, in MPS IIIA, gene therapy would involve the delivery of the SGSH gene, and in MPS IIIB, gene therapy would involve the delivery of the NAGLU gene. 3. Gene Editing: CRISPR-Cas9 and other gene editing technologies offer the possibility of directly correcting gene mutations. Using gene editing tools, the DNA sequence of mutated genes in affected cells can be precisely edited, either by correcting mutations or by replacing defective genes with normal copies. 4. Enhancement of cellular clearance mechanisms: Sanfilippo syndrome is characterized by the accumulation of GAGs in lysosomes due to a deficiency in enzyme activity. Gene therapy strategies may include enhancing cellular clearance mechanisms, such as autophagy or lysosomal function, to aid in the clearance of accumulated GAGs from cells. 5. Neuroprotective Factors: Sanfilippo syndrome affects the central nervous system and causes progressive neurological decline. Gene therapy techniques may include delivering neuroprotective factors or molecules that promote brain cell survival and function.

[0103] Potential gene therapies for Alzheimer's disease: Alzheimer's disease is the most common form of dementia and is characterized by amyloid plaques, cerebral amyloid angiopathy, and neurofibrillary tangles that affect the cerebral cortex, which is responsible for language, reason, and social behavior, as well as brain regions important for memory function, including the hippocampus.

[0104] Potential gene therapy strategies include the following: 1. Protective proteins / amyloid-degrading enzymes including cathepsin A and neprilysin, ECE, and cathepsin B. 2. Neurotrophic Factors: Gene therapy may involve delivering genes encoding neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), or glial cell line-derived neurotrophic factor (GDNF). These factors promote neuronal survival, growth, and function. 3. Antisense oligonucleotides that "silence" the tau protein gene. 4. siRNAs that downregulate Alzheimer's-related proteins such as BACE1 and APP. 5. Modification of APOE functionality.

[0105] Potential gene therapies for spinal cord injury: Treating spinal cord injury (SCI) with gene therapy is a complex undertaking due to the multifaceted nature of such injuries. However, several potential gene therapy approaches are being explored to promote nerve regeneration, reduce inflammation, and enhance functional recovery. Some of the transgenes that can be used are listed below. 1. Neurotrophic Factors: Gene therapy may involve delivering genes encoding neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), or glial cell line-derived neurotrophic factor (GDNF). These factors may enhance neuronal regeneration and functional recovery after SCI by promoting neuronal survival, growth, and function. 2. Anti-inflammatory factors: Inflammation plays a crucial role in the secondary injury mechanisms following SCI. Gene therapy techniques may aim to modulate the inflammatory response by delivering genes encoding anti-inflammatory factors such as interleukin-10 (IL-10), interleukin-1 receptor antagonists (IL-1RAs), or tumor necrosis factor alpha (TNF-α) inhibitors. 3. Extracellular Matrix Modulation: The extracellular matrix (ECM) provides structural support and guidance for axon growth. Gene therapy strategies may include delivering genes encoding ECM-modifying enzymes or proteins that promote ECM remodeling to facilitate axon regeneration and tissue repair. 4. Neural plasticity enhancers: Gene therapy techniques may aim to enhance neural plasticity and neural circuit rewiring after SCI. This may include delivering genes encoding proteins involved in synaptic plasticity, axon guidance, or dendritic spine formation. 5. Neuroprotective Factors: Gene therapy may involve delivering genes encoding neuroprotective factors or molecules that promote cell survival and function in the injured spinal cord. This can help mitigate secondary injury mechanisms and preserve neuronal function after SCI. 6. Cell replacement therapy: Gene therapy techniques may include delivering genes encoding factors that promote the differentiation and integration of transplanted stem cells or neural progenitor cells to the injured spinal cord, which may lead to functional recovery. 7. Modulation of Apoptosis: Gene therapy strategies may aim to modulate the apoptotic pathway to reduce post-SCI cell death and promote cell survival. This may involve delivering genes encoding anti-apoptotic factors or inhibitors of pro-apoptotic proteins.

[0106] Potential gene therapies for spinal muscular atrophy: Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the SMN1 gene, leading to a deficiency in survival motor neuron (SMN) protein. Gene therapy has emerged as a promising treatment for SMA, particularly by targeting the underlying genetic causes of the disease. The following are gene therapies used to treat various types of SMA. 1. SMA1 type: SMA1 type is the most severe form of the disease and typically appears in infancy. The main gene therapy drug used to treat SMA1 type is onasemnogene abeparvovec (brand name: Zolgensma). This is a one-time injection that delivers a functional copy of the SMN1 gene to replace the defective gene. Zolgensma uses an adeno-associated virus (AAV) vector to deliver the corrected gene to motor neurons. 2. SMA Type 2: SMA Type 2 usually manifests in infancy or early childhood, and the symptoms are less severe than those of Type 1. Zolgensma is primarily used for SMA Type 1, but its use in SMA Type 2 patients is also being explored. However, other therapeutic agents such as nusinersen (Spinraza) are more commonly used for SMA Type 2. Nusinersen is an antisense oligonucleotide that alters the splicing of SMN2 mRNA and increases the production of functional sex SMN protein. 3. SMA Type 3: SMA Type 3, also known as Kugelberg-Welander disease or juvenile SMA, typically appears later in childhood or adolescence and is milder than types 1 and 2. As of the inventors' previous update, no gene therapy drugs specifically for SMA Type 3 have been approved. Treatment for SMA Type 3 often involves multidisciplinary care, physiotherapy, and, in some cases, drug therapy to manage symptoms. However, ongoing research may lead to the development of gene therapy drugs targeting this type of SMA in the future.

[0107] Non-malignant CNS disorders can be selected from neurological disorders, including neurodegenerative diseases, enzyme deficiency conditions, neuroinflammatory diseases, acquired neurological injuries, or neuropsychiatric conditions. To avoid any doubt, non-malignant CNS disorders are not cancer.

[0108] Neurodegenerative diseases can be selected from dementia, Lewy body dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple system atrophy, spinal muscular atrophy, spinocerebellar ataxia, Friedreich's ataxia, Huntington's disease, Parkinson's disease, Parkinson's Plus syndrome, corticobasal degeneration, hypertrophic olivary degeneration, and leukodystrophy including Alexander disease, autosomal dominant leukodystrophy with autonomic nervous system disorders, Canavan disease, cerebral tendon xanthomatous disease, childhood ataxia with central nervous system myelin dysplasia, Krabbe disease, metachromatic leukodystrophy, Pelizaeus-Merzbach disease, Refsum's disease (adult or infant), Rett syndrome, and progressive supranuclear palsy.

[0109] The enzyme deficiency can be selected from lysosomal storage disorders, Tay-Sachs disease, Sandhoff disease, neurogenic ceroid lipofuscinosis, Hunter syndrome, Haller disease, Niemann-Pick disease, neuronal axonal dystrophy, Gaucher disease, and Sanfilippo syndrome (mucopolysaccharidosis type III).

[0110] Neuroinflammatory diseases can be selected from multiple sclerosis, neurosarcoidosis, encephalitis, prion diseases, and neuromyelitis optica (Devic's disease).

[0111] Acquired neurological injuries can be selected from stroke, traumatic brain injury, or spinal cord injury.

[0112] The neurological disorder may be epilepsy, essential tremor, or limbic encephalitis.

[0113] Gene or RNA therapeutic vectors are preferably sterile and can be administered in the form of a pharmaceutical composition which may contain one or more pharmaceutically acceptable carriers or excipients. Suitable carriers and excipients are well known to those skilled in the art and can be optimized depending on the intended delivery route. For example, suitable pharmaceutical compositions may include buffers, binders, preservatives, thickeners, or antioxidants.

[0114] The gene or RNA therapy vector is preferably intended for administration by infusion over 1 to 24 hours, particularly at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours, and / or less than 10, 9, 8, 7, 6, 5, 4, or 3 hours. Infusions of about 5 hours or about 3 hours are preferred. In embodiments of the present invention, the gene or RNA therapy vector can be repeatedly infused daily for up to 6 days.

[0115] Whether the gene or RNA therapy vector is administered over several consecutive days or periodically over several days, the gene or RNA therapy vector may be administered independently or additionally weekly, every two weeks, monthly, every six weeks, every eight weeks, every twelve weeks, or every fifteen weeks, or more frequently. For example, a two or three-day infusion cycle may be repeated every three months. Alternatively, it may be administered in a series of infusion cycles with an interval of six, seven, eight, nine, ten, eleven, or twelve months between the completion of the first infusion cycle and the next.

[0116] Non-malignant CNS disorders are preferably treated in subjects that require it. Subjects are preferably mammals, more preferably primates, and especially humans, and may be pediatric or elderly patients.

[0117] Furthermore, the present invention provides a method for treating non-malignant CNS disorders, comprising injecting a gene or RNA therapeutic vector into the pons by CED according to the embodiments of the present invention described above.

[0118] The present invention will be described in detail below with reference to the drawings, although these are merely illustrative examples. [Brief explanation of the drawing]

[0119] [Figure 1]This figure shows an axial diagram (A) and a histological specimen (B) passing through the pons. This figure shows the structure within the pons at the axial level of the middle cerebellar peduncle. Anterior to this level are the descending motor corticospinal tract and the corticobulbar tract. Posterior to these are the transverse pontine (pontoon-cerebellar) fibers that connect the cerebral cortex to the contralateral cerebellar cortex via the middle cerebellar peduncle. Further posterior to these is the medial lemniscus, which contains secondary neurons of the posterior sensory column that play an important role in proprioception, vibration detection, fine touch, and two-point discrimination. Throughout the pons, there are important nuclei that serve as origins for cranial nerves. [Figure 2] Figure 2B is an axial diagram of the pons, middle cerebellar peduncle, and deep cerebellar nuclei. The pons is connected to the cerebellum via the middle cerebellar peduncle. At this level are the deep cerebellar nuclei (DCNs), which play a crucial role in the transmission of motor fibers from the cerebellum to the thalamus and motor cortex to optimize motor precision and coordination. Note: 1. Pons, 2. Middle cerebellar peduncle, 3. Tubercle, 4. Fourth ventricle, 5. Deep cerebellar nuclei (DCNs). Figure 2B shows an axial diagram, where the pons and middle cerebellar peduncle are at a more caudal level. This illustrates the relationship between the inferior cerebellar peduncle and the middle cerebellar peduncle in this region. Infusion of gene therapy drugs covering the middle cerebellar peduncle would also distribute to the inferior cerebellar peduncle, which contains spinocerebellar fibers, inferior olivocerebellar fibers, and vestibular cerebellar fibers, which are thought not to be covered by infusions limited to the pons and transmit information essential for coordinated motor function and learning. [Figure 3] This is an illustrative diagram of bilateral transcerebellar cannula placement from the middle cerebellar peduncle. The cannula step is positioned at the interface between the DCN(5) and the posterior white matter. The cannula trajectory crosses the side of the DCN rather than injecting directly into the DCN to prevent damage to these important nuclei (A). Figure 3B is the corresponding axial MRI image after unilateral cannula injection of AAV9-mCherry along the left middle cerebellar peduncle and pons with co-injection of MRI contrast. (B) shows the sagittal view of the trajectory and injection. [Figure 4]This is a schematic diagram illustrating the relationship between the DCN, Purkinje cells, and the inferior olivary nucleus. The dentate nucleus is the largest part of the DCN. Purkinje cells in the cerebellar cortex project their axons through the motor thalamus to the dentate nucleus, inhibiting their movement into the motor cortex. The inferior olivary nucleus is a source of climbing fibers for Purkinje cells in the cerebellar cortex. These structures play a crucial role in motor coordination. [Figure 5] The axial MR images before (left) and after (center) injection, as well as the sagittal plane image (right), confirm the bilateral distribution of gadolinium in the pons. [Figure 6] When GFP expression is measured by ELISA, expression of AAV5-GFP is confirmed in the ipsilateral motor cortex, contralateral motor cortex, and spinal cord one month after unilateral pontine injection. [Figure 7] When mCherry expression is measured by ELISA, expression is confirmed in the ipsilateral motor cortex, contralateral motor cortex, and spinal cord one month after unilateral pontine injection of AAV9-mCherry. [Figure 8] Fluorescence microscopy confirms mCherry expression in the sublingual nucleus and the inferior olivary nucleus of the medulla. [Figure 9] Fluorescence microscopy confirms the expression of mCherry in anterior horn cells of the spinal cord. [Figure 10] When mCherry expression is measured in pg / ml units by ELISA after unilateral (left pons) injection, expression is confirmed in the contralateral and ipsilateral submotor systems—orbitofrontal cortex, motor cortex, premotor cortex, caudate nucleus, putamen, motor thalamus, pons, trigeminal nerve origin, vestibular nuclei, hypoglossal nucleus, cerebellar cortex, dentate nucleus, and anterior horn cells of the spinal cord. Samples exceeding the upper detection threshold (logarithmic scale) are assigned an arbitrary value of 400,000. [Figure 11] When mCherry expression is measured in pg / ml units by ELISA after unilateral (left pons) injection, expression is confirmed in the contralateral and ipsilateral sensory subsystems—sensory cortex, auditory cortex, sensory thalamus, Clark column, and posterior column of the spinal cord. Samples exceeding the upper detection threshold (logarithmic scale) are assigned an arbitrary value of 400,000. [Figure 12]When mCherry expression is measured in pg / ml units by ELISA after unilateral (left pons) injection, expression is confirmed in the contralateral and ipsilateral subsystems of the visual system—optic tract, visual cortex, and visual association cortex. Samples exceeding the upper detection threshold (logarithmic scale) are assigned an arbitrary value of 400,000. [Figure 13] When mCherry expression is measured in pg / ml units by ELISA after injection into one side (left pons), expression is confirmed in the contralateral and ipsilateral peripheral nerves and muscles—sciatic nerve, tibial nerve, spinal dorsal root ganglia, buccinator muscle, and tongue. [Figure 14] When mCherry expression is measured in pg / ml units by ELISA after unilateral (left pons) injection, expression is confirmed in the contralateral and ipsilateral lower limbic system—amygdala, hippocampus, and temporal lobe cortex. [Figure 15] Detection of mCherry by DAB immunohistochemistry reveals its expression in the bilateral orbitofrontal cortex and anterior frontal cortex. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 16] Detection of mCherry by DAB immunohistochemistry confirms its expression in the left and right primary motor cortex and somatosensory cortex. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 17] Detection of mCherry by DAB immunohistochemistry revealed its expression in the left and right caudate nuclei and putamen. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 18] Detection of mCherry by DAB immunohistochemistry reveals its expression in the left and right temporal cortex, parietal cortex, hypothalamus, and amygdala. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 19]Detection of mCherry by DAB immunohistochemistry reveals its expression in the lateral geniculate nucleus, optic tract, auditory cortex, parietal cortex, hippocampus, medial lemniscus, and thalamus. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 20] Detection of mCherry by DAB immunohistochemistry revealed its expression in the left and right pons, superior colliculus, raphe nuclei, visual cortex, and visual association cortex. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 21] Detection of mCherry by DAB immunohistochemistry revealed its expression in the left and right cerebellar cortex, deep cerebellar nuclei, vestibular nuclei, inferior cerebellar peduncle, and pyramidal region. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 22] Detection of mCherry by DAB immunohistochemistry reveals its expression in the medulla, cuneate nucleus, gracile nucleus, vagus nucleus, inferior olivary nucleus, pyramidal nucleus, medial longitudinal nucleus, and trigeminal nucleus. Histology (right image is shown with the corresponding section from the Wisconsin Sheep Brain Atlas (left image)). [Figure 23] When mCherry is detected by DAB immunohistochemistry, its expression is confirmed in Clark's column in the cervical spinal cord (left image), anterior horn cells in the thoracic spinal cord (center image), and anterior horn cells in the lumbar spinal cord (right image). [Examples]

[0120] 1. Injection of AAV9-mCherry and AAV5-GFP All work was carried out under the authority of the Animals (Scientific Treatment) Act (1986) and the appropriate UK Home Office project and personal licenses. The sheep model is particularly relevant because the development of projections from the brainstem nuclei is the same as in other mammalian species (Stockx et al., 2007).

[0121] Sheep anesthesia The subjects were moved to the induction room and ketamine (10 mg / kg) and dexmedetomidine (15 mcg / kg) were administered intramuscularly to the upper neck. An intravenous (IV) catheter was placed in the ear vein. Induction of general anesthesia was achieved by intravenous administration of propofol at a 1.0 mg / kg bolus until the eyelid reflex disappeared. After induction, the airway was secured with an appropriately sized endotracheal tube. Anesthesia was maintained with 1–2.5% isoflurane in oxygen. The animals were subjected to mechanical ventilation with target values ​​of 10–20 ml / kg Vt and 10–20 breaths / min RR. Settings were adjusted to maintain ET-CO2 at 35–45 mmHg. Before proceeding with the experiment, the animals were secured to the operating table and positioned in the sternal position.

[0122] To prepare the surgical field, precise shaving and cleansing of the skin in the cervical (or groin) region were achieved. Monitoring devices included: • Invasive arterial blood pressure catheter • Peripheral oxygen saturation probe • Deep temperature probe • Central venous line inserted into the jugular vein

[0123] Surgical preparation and preoperative preparation: The subject's skull was fixed using a custom-made sheep model fixation frame in the preparation room and throughout the imaging, surgical, and injection procedures. Anatomical location was performed using a dedicated reference frame and integrated co-registration module mounted on the fixation frame before transferring to the MRI scanner.

[0124] MRI scan Surgical planning and real-time continuous scanning of intracranial injections were performed using a 3T MRI unit (Prisma, Siemens). Surface receiving coils were used in combination with sheep fixation and a reference platform.

[0125] Detailed trajectory planning and surgery Once the MRI imaging was complete, the patient was moved to the operating room for the implantation of bilateral perfusion cannulas into the pons.

[0126] After MRI imaging, the anesthetized sheep was moved to the operating room and positioned on the operating table for bilateral insertion of cannulas into the pons. The head was prepared and draped, and the CRW stereotactic guide was set to the coordinates of the first target. Through a 10-20 mm linear scalp incision, a contour hole coaxial with the trajectory was drilled into the skull using custom tools and setting jigs. The guide hub was delivered into the contour hole and secured with a self-tapping screw. A guide tube cut to length was inserted through the guide hub, followed by a priming cannula cut to length. The priming cannula was secured to the hub by a screw. The cannula contained a continuous 2-meter length PEEK tube (0.5 mm OD, 0.127 mm ID) with a screw at 5 cm from the distal end and connected to a 500 μl Hamilton® syringe at the proximal end. The dead volume of the catheter and infusion line was 25 μl.

[0127] Next, the CRW coordinates of the second target were set. The second cannula was inserted and secured at this point as described above.

[0128] injection Injection of AAV vectors into aCSF containing 0.2% gadolinium was performed as shown in Table 1. Reporter genes mCherry and GFP were selected to enable analysis by ELISA, fluorescence microscopy, and DAB immunohistochemistry.

[0129] [Table 1]

[0130] analysis An MRI scan was performed immediately after injection to determine the distribution of gadolinium. The subjects showed no adverse neurological, respiratory, or cardiac signs throughout the surgical, injection, and recovery periods. One month after injection, the subjects were sacrificed, and their brains and spinal cords were removed. The brains and spinal cords removed from subject 1 were immediately frozen for ELISA analysis of mCherry and GFP expression. The brains removed from subject 2 were formalin-fixed for fluorescence microscopy.

[0131] The brains excised from Subject 3 were cut into 0.5 cm coronal sections. Alternating sections showing mCherry expression by ELISA were immediately frozen. The remaining sections were formalin-fixed for DAB immunohistochemistry. The spinal cord was cut into 5 cm sections. The proximal 1 cm of each section was frozen for ELISA, and the remaining 4 cm was formalin-fixed for DAB immunohistochemistry.

[0132] Results - MRI after injection Immediately after injection, MRI confirmed that gadolinium was distributed throughout the pons (Figure 6).

[0133] Results - GFP ELISA: Subjects 1 & 2 One month after injecting AAV5-GFP into the left pons, GFP expression was identified in the ipsilateral motor cortex, contralateral motor cortex, and spinal cord (Figure 6).

[0134] Results - mCherry ELISA: Subjects 1 & 2 One month after injecting AAV9-mCherry into the right pons, mCherry expression was identified in the ipsilateral motor cortex, contralateral motor cortex, and spinal cord (Figure 7).

[0135] Results - mCherry fluorescence microscopy mCherry was visualized in the medulla and sublingual nucleus within the inferior olivary nucleus (Figure 8) and in the spinal cord (Figure 9).

[0136] Results - mCherry ELISA: Subject 3 (unilateral pontine injection) One month after injecting AAV9-mCherry into the left pons, mCherry expression was identified bilaterally in the following areas. - Orbitofrontal cortex, premotor and motor cortex, putamen, caudate nucleus, motor thalamus, vermis, origin of the trigeminal nerve, cerebellar cortex, dentate nucleus, and anterior horn cells of the cervical, thoracic, and lumbar spinal cord (Figure 10). - Sensory cortex, auditory cortex, Clark column, and posterior column (Figure 11). - The optic tract, visual cortex, and visual association cortex (Figure 12). - Sciatic nerve, tibial nerve, spinal dorsal root ganglion, buccinator muscle (right side only), and tongue muscle (Figure 13). - Hippocampus, amygdala, and temporal cortex (Figure 14).

[0137] Results - mCherry DAB immunohistochemistry: Subject 3 (unilateral pontine injection) One month after injecting AAV9-mCherry into the left pons, mCherry expression was detected bilaterally in the following areas. - Orbitofrontal cortex and prefrontal cortex (Figure 15). - Motor cortex and somatosensory cortex (Figure 16). - Caudate nucleus and putamen (Figure 17). - Temporal and parietal cortex, thalamus, hypothalamus, and amygdala (Figure 18). - Lateral geniculate nucleus, optic tract, auditory cortex, parietal cortex, hippocampus, medial lemniscus, and thalamus (Figure 19). - Pons, superior colliculus, raphe nuclei, visual cortex, and visual association cortex (Figure 20). - Cerebellar cortex, deep cerebellar nuclei, vestibular nuclei, inferior cerebellar peduncle, and pyramidal region (Figure 21). - Medulla, cuneate nucleus and gracile nucleus, vagus nucleus, inferior olivary nucleus, pyramidal nucleus, medial longitudinal nucleus, and trigeminal spinal nucleus (Figure 22). - Clark's column, as well as anterior horn cells of the cervical, thoracic, and lumbar spinal cord (Figure 23).

[0138] conclusion These data demonstrate that a single CED injection of an adeno-associated virus vector into the pons via a transcerebellar orbit leads to gene expression in multiple components of the cerebral cortex, motor system, sensory system, and limbic system necessary for the modulation of motor activity—including the motor cortex, basal ganglia, sensory cortex, auditory cortex, visual cortex, and visual association cortex, hippocampus and amygdala, spinal cord, cranial nerve nuclei, inferior olivary nucleus, thalamus, dentate nucleus, cerebellum, and anterior horn cells of the spinal cord, as well as in the peripheral nervous system and muscles.

[0139] References Kimura S & Harashima H., Pharmaceutics. 2020; 15;12(12):1216 Bailey RM et al., Mol Ther Methods Clin Dev. 2018; 15;9:160-171 Kells AP et al., Proc Natl Acad Sci USA. 2009; 106(7):2407-2411 Khateeb K et al., J Vis Exp. 2019; 23;(147) Nieuwenhuis B et al., Gene Therapy 2021; 28:56-74 Barua NU et al., J Neurosci Methods. 2013; 220(1):1-8 Salegio EA et al., Hum Gene Ther. 2022; 33(1-2): 86-93 Bravo-Hernandez et al., Nat Med. 2020; 26(1):118-130 Salegio et al., Mol Ther. 2010; 18(8):1490-1495 Li et al., Neurosci. Bull. 2019; 35(5):909-920 Gill T, et al. J Neurosci Methods. 2013; 219(1):1-9 Panse SJ, et al. J Med Eng Technol. 2010; 35(7-8):408-414. Arrigo et al. Neuroscience. 2017; 343, P346-354. McLachlan & Wilson. Fron Psychol. 2017; 8:265. Stockx et al. BMC Neurosci. 2007; 8:40.

Claims

1. A gene or RNA therapy vector for use in the treatment of non-malignant CNS disorders, which is to be injected into the pons by convection-enhanced delivery (CED).

2. A gene or RNA therapeutic vector for use according to claim 1, which will be injected into Maebashi.

3. A gene or RNA therapeutic vector for use according to claim 1 or 2, which will also be injected into the middle cerebellar peduncle.

4. The injection is performed from at least one cannula placed via a posterior-to-anterior transcerebellar orbit that transversely crosses to the deep cerebellar nuclei, wherein the gene or RNA therapeutic vector for use according to any one of claims 1 to 3.

5. The cannula is a stepped cannula, a gene or RNA therapeutic vector for use according to any one of claims 1 to 4.

6. The cannula step is positioned at the interface between the posterior deep cerebellar nuclei and the cerebellar white matter, the gene or RNA therapeutic vector for use according to claim 5.

7. The gene or RNA therapy vector for use according to any one of claims 1 to 6, wherein the gene or RNA therapy vector is injected into the pons to deliver the gene or RNA therapy vector by anterograde and / or retrograde transport to one or more of the cerebral cortex, basal ganglia, thalamus, visual system, auditory system, and limbic system, ascending and descending sensory and motor pathways, brainstem nuclei, deep cerebellar nuclei, cerebellar white matter, cerebellar Purkinje cells, inferior olivary nucleus, peripheral nerves, and muscles.

8. A gene or RNA therapeutic vector for use according to any one of claims 1 to 7, which is a virus or a non-viral vector.

9. A gene or RNA therapeutic vector for use according to claim 8, which is a viral vector selected from adenovirus, adeno-associated virus (AAV), lentivirus, and herpesvirus.

10. A gene or RNA therapeutic vector for use according to claim 8, which is AAV5 or AAV9.

11. The aforementioned viral vector is 10 ５ ~10 １５ A gene or RNA therapeutic vector for use according to claim 9 or 10, administered at a concentration of Vg / ml.

12. A gene or RNA therapeutic vector for use according to claim 8, which is a nonviral vector selected from polymers, lipids, peptides, inorganic substances, and hybrid systems.

13. The non-malignant CNS disorder is selected from neurodegenerative diseases, neurological disorders, enzyme deficiency conditions, neuroinflammatory diseases, acquired neurological injuries, or neurological disorders, and is a gene or RNA therapeutic vector for use according to any one of claims 1 to 12.

14. The neurological disease is selected from diseases characterized primarily by pyramidal degeneration, including amyotrophic lateral sclerosis, spinal muscular atrophy, spinal muscular atrophy type 1 with respiratory distress, congenital SMA with joint contracture, progressive bulbar palsy, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease, and post-polio syndrome, and is a gene or RNA therapy vector for use according to claim 13.

15. The gene or RNA therapeutic vector for use according to claim 13, wherein the neurological disorder is selected from disorders characterized primarily by degeneration of extrapyramidal motor pathways, including Parkinson's disease and Parkinson's disease plus syndrome, Huntington's disease, dystonia, hemiballism, Tourette syndrome, Sydenham chorea, cerebral palsy with motor dysfunction, Athymformic syndrome, Lesch-Nyhan syndrome, and Wilson's disease.

16. The gene or RNA therapeutic vector for use according to claim 13, wherein the neurological disorder is selected from disorders characterized primarily by degeneration of the cerebral cortex, including Alzheimer's disease, frontotemporal dementia, Creutzfeldt-Jakob disease, and other forms of dementia.

17. The gene or RNA therapeutic vector for use according to claim 13, wherein the neurological disorder is selected from disorders characterized by marked degeneration of the cerebellar system, including Friedreich's ataxia and spinocerebellar ataxia.

18. The gene or RNA therapeutic vector for use according to claim 13, wherein the neurological disorder is selected from disorders characterized by limbic system disorders, including epilepsy, limbic encephalitis, dementia, anxiety disorders, schizophrenia, and autism spectrum disorder.

19. The neurological disorder is selected from a range of disorders characterized by widespread pathological changes throughout the nervous system, including leukodystrophy including Lewy body disease, multiple system atrophy, corticobasal degeneration, hypertrophic olivary degeneration, and Alexander disease; autosomal dominant leukodystrophy with autonomic nervous system disorder; Canavan disease; cerebral tendon xanthomatous disease; childhood ataxia with central nervous system myelin dysplasia; Krabbe disease; heterochromatic leukodystrophy; Pelizaeus-Merzbach disease; Refsum's disease (adult or infant); Rett syndrome and progressive supranuclear palsy; myelin dysplasia with basal ganglia and cerebellar atrophy; and essential tremor, a gene or RNA therapeutic vector for use according to claim 13.

20. The gene or RNA therapeutic vector for use according to claim 13, wherein the neurological disorder is selected from lysosomal storage disorders, Tay-Sachs disease, Sandhoff disease, neuronal ceroid lipofuscinosis including Batten disease, Hunter syndrome, Haller disease, Niemann-Pick disease, neuronal axonal dystrophy, Gaucher disease, and enzyme deficiency disorders including Sanfilippo syndrome (mucopolysaccharidosis type III).

21. The gene or RNA therapeutic vector for use according to claim 13, wherein the neurological disorder is selected from neuroinflammatory disorders including multiple sclerosis, neurosarcoidosis, encephalitis, prion diseases, and neuromyelitis optica (Devic's disease).

22. The gene or RNA therapeutic vector for use according to claim 13, wherein the neurological disorder is selected from neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD), affective disorders, Asperger's syndrome, and addiction.

23. The gene or RNA therapeutic vector for use according to claim 13, wherein the acquired neurological injury is a stroke, traumatic brain injury, or spinal cord injury.

24. A method for treating a non-malignant CNS disorder, comprising injecting a gene or RNA therapeutic vector into the pons of a subject requiring the treatment (CED).

25. The method according to claim 24, wherein the gene or RNA therapy vector is injected into Maebashi.

26. The method according to claim 24 or 25, wherein the gene or RNA therapy vector is also injected into the middle cerebellar peduncle.

27. The method according to any one of claims 24 to 26, wherein the injection is performed from at least one cannula placed via a transcerebellar trajectory from posterior to anterior that transversely crosses to the deep cerebellar nuclei.

28. The method according to any one of claims 24 to 27, wherein the cannula is a stepped cannula.

29. The method according to claim 28, wherein the cannula step is positioned at the interface between the posterior deep cerebellar nuclei and the cerebellar white matter.

30. The method according to any one of claims 24 to 29, wherein the gene or RNA therapeutic vector is injected into the pons to deliver the gene or RNA therapeutic vector by anterograde and / or retrograde transport to one or more of the ascending and descending sensory and motor pathways, brainstem nuclei, deep cerebellar nuclei, cerebellar white matter, cerebellar Purkinje cells, and inferior olivary nucleus.

31. The method according to any one of claims 24 to 30, wherein the gene or RNA therapeutic vector is a virus or a non-viral vector.

32. The method according to claim 31, wherein the gene or RNA therapeutic vector is a viral vector selected from adenoviruses, adeno-associated viruses (AAVs), lentiviruses, and herpesviruses.

33. The method according to claim 32, wherein the gene or RNA therapy vector is AAV5 or AAV9.

34. The aforementioned viral vector is 10 ５ ~10 １５ The method according to claim 32 or 33, administered at a concentration of Vg / ml.

35. The method according to claim 31, wherein the gene or RNA therapeutic vector is a nonviral vector selected from polymers, lipids, peptides, inorganic substances, and hybrid systems.

36. The method according to any one of claims 24 to 35, wherein the non-malignant CNS disorder is selected from neurodegenerative diseases, neurological diseases, enzyme deficiency conditions, neuroinflammatory diseases, acquired neurological injuries, or neurological disorders.

37. The method according to claim 36, wherein the neurological disease is selected from diseases characterized primarily by degeneration of pyramidal motion, including amyotrophic lateral sclerosis, spinal muscular atrophy, spinal muscular atrophy type 1 with respiratory distress, congenital SMA with joint contracture, progressive bulbar palsy, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease, and post-polio syndrome.

38. The method according to claim 36, wherein the neurological disorder is selected from disorders mainly characterized by degeneration of the extrapyramidal motor pathway, including Parkinson's disease and Parkinson's disease plus syndrome, Huntington's disease, dystonia, hemiballism, Tourette syndrome, Sydenham chorea, cerebral palsy with motor dysfunction, Athymformic syndrome, Lesch-Nyhan syndrome, and Wilson's disease.

39. The method according to claim 36, wherein the neurological disorder is selected from disorders characterized primarily by degeneration of the cerebral cortex, including Alzheimer's disease, frontotemporal dementia, Creutzfeldt-Jakob disease, and other forms of dementia.

40. The method according to claim 36, wherein the neurological disorder is selected from disorders characterized by marked degeneration of the cerebellar system, including Friedreich's ataxia and spinocerebellar ataxia.

41. The method according to claim 36, wherein the neurological disorder is selected from disorders characterized by limbic system disorders, including epilepsy, limbic encephalitis, dementia, anxiety disorders, schizophrenia, and autism spectrum disorder.

42. The method according to claim 36, wherein the neurological disorder is selected from a disorder characterized by widespread pathological changes throughout the nervous system, including leukodystrophy including Lewy body disease, multiple system atrophy, corticobasal degeneration, hypertrophic olivary degeneration, and Alexander disease, autosomal dominant leukodystrophy with autonomic nervous system disorder, Canavan disease, cerebral tendon xanthomatous disease, childhood ataxia with central nervous system myelin dysplasia, Krabbe disease, metachromatic leukodystrophy, Pelizaeus-Merzbach disease, Refsum's disease (adult or infant), Rett syndrome and progressive supranuclear palsy, myelin dysplasia with basal ganglia and cerebellar atrophy, and essential tremor.

43. The method according to claim 36, wherein the neurological disorder is selected from lysosomal storage disorders, Tay-Sachs disease, Sandhoff disease, neurogenic ceroid lipofuscinosis including Batten disease, Hunter syndrome, Haller disease, Niemann-Pick disease, neuronal axonal dystrophy, Gaucher disease, and enzyme deficiency disorders including Sanfilippo syndrome (mucopolysaccharidosis type III).

44. The method according to claim 36, wherein the neurological disorder is selected from neuroinflammatory disorders including multiple sclerosis, neurosarcoidosis, encephalitis, prion disease, and neuromyelitis optica (Devic's disease).

45. The method according to claim 36, wherein the neurological disorder is selected from neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD), affective disorders, Asperger's syndrome, and addiction.

46. The method according to claim 36, wherein the acquired neurological injury is a stroke, traumatic brain injury, or spinal cord injury.

47. A method for distributing a gene therapy drug throughout the nervous system, wherein a viral vector, which is transported anterograde and retrogradely by axons, is injected into the target pons and pons from a cannula inserted from the cerebellum and cerebellar peduncle into the pons.

48. The method according to claim 47, wherein the distribution throughout the nervous system includes the frontal, temporal, parietal, and occipital cerebral cortex.

49. The method according to claim 47 or 48, wherein the distribution throughout the nervous system includes subcortical brain structures including the thalamus, basal ganglia, hippocampus, and amygdala.

50. The method according to any one of claims 47 to 49, wherein the distribution throughout the nervous system includes the brainstem, spinal cord, and peripheral nerves.

51. The method according to any one of claims 47 to 50, wherein the distribution throughout the nervous system includes the cerebellum and deep cerebellar nuclei.

52. The method according to any one of claims 47 to 51, wherein the distribution throughout the nervous system includes a visual pathway comprising the optic tract, lateral geniculate nucleus, superior colliculus, visual cortex, and visual association area.

53. The method according to any one of claims 47 to 52, wherein the viral vector is selected from adenovirus, adeno-associated virus (AAV), lentivirus, and herpesvirus.

54. The method according to claim 53, wherein the AV is AAV5 or AAV9.

55. The method according to any one of claims 47 to 54, wherein the gene therapy agent is for treating non-malignant CNS disorders.

56. The method according to claim 55, wherein the non-malignant CNS disorder is selected from neurodegenerative diseases, neurological diseases, enzyme deficiency conditions, neuroinflammatory diseases, acquired neurological injuries, or neurological disorders.

57. The method according to claim 56, wherein the neurodegenerative disease is selected from diseases characterized primarily by pyramidal degeneration, including amyotrophic lateral sclerosis, spinal muscular atrophy, spinal muscular atrophy type 1 with respiratory distress, congenital SMA with joint contracture, progressive bulbar palsy, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease, and post-polio syndrome.

58. The method according to claim 56, wherein the neurological disorder is selected from disorders mainly characterized by degeneration of the extrapyramidal motor pathway, including Parkinson's disease and Parkinson's disease plus syndrome, Huntington's disease, dystonia, hemiballism, Tourette syndrome, Sydenham chorea, cerebral palsy with motor dysfunction, Athymformic syndrome, Lesch-Nyhan syndrome, and Wilson's disease.

59. The method according to claim 56, wherein the neurological disorder is selected from disorders characterized primarily by degeneration of the cerebral cortex, including Alzheimer's disease, frontotemporal dementia, Creutzfeldt-Jakob disease, and other forms of dementia.

60. The method according to claim 56, wherein the neurological disorder is selected from disorders characterized by marked degeneration of the cerebellar system, including Friedreich's ataxia and spinocerebellar ataxia.

61. The method according to claim 56, wherein the neurological disorder is selected from disorders characterized by limbic disorders, including epilepsy, limbic encephalitis, dementia, anxiety disorders, schizophrenia, and autism spectrum disorder.

62. The method according to claim 56, wherein the neurological disorder is selected from a disorder characterized by widespread pathological changes throughout the nervous system, including leukodystrophy including Lewy body disease, multiple system atrophy, corticobasal degeneration, hypertrophic olivary degeneration, and Alexander disease, autosomal dominant leukodystrophy with autonomic nervous system disorder, Canavan disease, cerebral tendon xanthomatous disease, childhood ataxia with central nervous system myelin dysplasia, Krabbe disease, metachromatic leukodystrophy, Pelizaeus-Merzbach disease, Refsum's disease (adult or infant), Rett syndrome and progressive supranuclear palsy, myelin dysplasia with basal ganglia and cerebellar atrophy, and essential tremor.

63. The method according to claim 56, wherein the neurological disorder is selected from lysosomal storage disorders, Taysachs disease, Sandhoff disease, neurogenic ceroid lipofuscinosis including Batten disease, Hunter syndrome, Haller disease, Niemann-Pick disease, neuronal axonal dystrophy, Gaucher disease, and enzyme deficiency disorders including Sanfilippo syndrome (mucopolysaccharidosis type III).

64. The method according to claim 56, wherein the neurological disorder is selected from neuroinflammatory disorders including multiple sclerosis, neurosarcoidosis, encephalitis, prion diseases, and neuromyelitis optica (Devic's disease).

65. The method according to claim 56, wherein the neurological disorder is selected from neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD), affective disorders, Asperger's syndrome, and addiction.

66. The method according to claim 56, wherein the enzyme deficiency is selected from lysosomal storage disorders, Tay-Sachs disease, Sandhoff disease, neurogenic ceroid lipofuscinosis, Hunter syndrome, Haller disease, Niemann-Pick disease, neuronal axonal dystrophy, and Gaucher disease.

67. The method according to claim 56, wherein the neuroinflammatory disease is selected from multiple sclerosis, neurosarcoidosis, encephalitis, and prion disease.

68. The method according to claim 56, wherein the acquired neurological injury is a stroke, traumatic brain injury, or spinal cord injury.

69. The method according to claim 56, wherein the neurological disorder is epilepsy or essential tremor.

70. The method according to any one of claims 47 to 69, wherein the cannula is a reflux-resistant cannula such as a stepped cannula.

71. The method according to any one of claims 47 to 70, wherein the transcerebellar peduncle injection is unilateral.

72. The method according to any one of claims 47 to 70, wherein the transcerebellar peduncle injection is bilateral.

73. The method according to any one of claims 47 to 72, wherein the injection further comprises an injection from a cannula inserted unilaterally or bilaterally into the pons via the cerebral peduncle.

74. The method according to any one of claims 47 to 73, wherein the injection volume is 50 μl to 10 ml per day.

75. The method according to any one of claims 47 to 74, wherein the injection flow rate is 0.5 μL / min to 20 μL / min.

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