Gene Delivery and Therapy for Neurological Disorders

Unique gene therapy prevents, reverses multiple sclerosis in animal model
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For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e. The Rep78 proteins of various AAV serotypes are e. Virol, 73 2 The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid proteins of one serotype e.

Such pseudotyped rAAV particles are a part of the present invention. Modified "AAV" sequences also can be used in the context of the present invention, e. Such modified sequences e. In view thereof, the production of rAAV5 can differ from production of other serotypes in insect cells.

Where methods of the invention are employed to produce rAAV5, it is preferred that one or more constructs comprise, collectively in the case of more than one construct, a nucleotide sequence comprising an AAV5 ITR, a nucleotide sequence comprises an AAV5 Rep coding sequence i. Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell , J. Data Standards Manual. Food and Drug Administration. Retrieved 11 March This is an injection through the theca of the spinal cord into the subarachnoid space so that it reaches the CSF.

This method of administration is useful in for example spinal anaesthesia, chemotherapy, or pain management applications. This route is also used to introduce drugs that fight certain infections, particularly post-neurosurgical. A substance administered via intrathecal injection avoids the need to passage the blood brain barrier. Drugs given intrathecally often do not contain any preservative or other potentially harmful inactive ingredients that are sometimes found in medicaments for intravenous injection.

Characteristics of foamy virus (Figure 1)

The lumbar facet joints allow for significant flexion and extension movement but limits rotation. For our vectorized antibody programs targeting tau and alpha-synuclein, we are pursuing an intravenous route of administration. Figure As there are two promoters present in the genome of foamy virus, it is presumed that IP functionally replaces the post-transcriptional regulatory protein of other retroviruses and thus provides a temporal pattern with stage-specific promoters Mergia, Browse All Figures Return to Figure. Country of ref document : EA.

The intrathecal method of delivery is regarded as less invasive than infusion into the central nervous system CNS tissue itself, because infusion into the CNS requires complex brain surgery. Intrathecal delivery can be performed without the need of a specialized center for brain injections. The neck region of the spine is known as the Cervical Spine. This region consists of seven vertebrae, which are abbreviated CI through C7 top to bottom. These vertebrae protect the brain stem and the spinal cord, support the skull, and allow for a wide range of head movement.

The first cervical vertebra CI is called the Atlas. The Atlas is ring-shaped and it supports the skull. C2 is called the Axis. It is circular in shape with a blunt tooth-like structure called the Odontoid Process or dens that projects upward into the Atlas. Together, the Atlas and Axis enable the head to rotate and turn.

The other cervical vertebrae C3 through C7 are shaped like boxes with small spinous processes finger-like projections that extend from the back of the vertebrae. Beneath the last cervical vertebra are the 12 vertebrae of the Thoracic Spine. These are abbreviated Tl through T12 top to bottom. Tl is the smallest and T12 is the largest thoracic vertebra. The thoracic vertebrae are larger than the cervical bones and have longer spinous processes. In addition to longer spinous processes, rib attachments add to the thoracic spine's strength. These structures make the thoracic spine more stable than the cervical or lumbar regions.

In addition, the rib cage and ligament system limit the thoracic spine's range of motion and protects many vital organs. The size and shape of each lumbar vertebra is designed to carry most of the body's weight. Each structural element of a lumbar vertebra is bigger, wider and broader than similar components in the cervical and thoracic regions.

The lumbar spine has more range of motion than the thoracic spine, but less than the cervical spine. The lumbar facet joints allow for significant flexion and extension movement but limits rotation. The Sacrum is located behind the pelvis. Five bones, abbreviated SI through S5, fused into a triangular shape, form the sacrum. The sacrum fits between the two hip bones connecting the spine to the pelvis. The last lumbar vertebra L5 articulates moves with the sacrum.

Immediately below the sacrum are five additional bones, fused together to form the Coccyx tailbone. In a preferred embodiment of the present invention, the gene therapy vector is administered by lumbar administration. Most preferably, the gene therapy vector is administered by intrathecal injection between L4 and L5. In one preferred embodiment, the gene therapy vector is administrated only by lumbar administration, i.

Alternatively or in combination with a previously preferred embodiment of the present invention, in a preferred embodiment of the invention the gene therapy vector is administered into the cisterna magna. In one preferred embodiment, the gene therapy vector is administrated only into the cisterna magna, i. More preferably, administration into the cisterna magna is performed in combination with the intrathecal administration, or in other words prior to, simultaneous or after the intrathecal administration.

In another preferred embodiment, administration into the cisterna magna is performed in combination with the lumbar administration, whereby preferably administration into the cisterna magna is performed prior to, simultaneous or after the lumbar administration. Preferably in this embodiment, the lumbar administration is in the intrathecal region. In a most preferred embodiment, the gene therapy vector is administered only into the cisterna magna in combination with the lumbar administration, i. The "cisterna magna" or "cerebellomedullary cistern" is one of three principal openings in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain.

The openings are collectively referred to as cisterns. The cisterna magna is located between the cerebellum and the dorsal surface of the medulla oblongata. Cerebrospinal fluid produced in the fourth ventricle drains into the cisterna magna via the lateral apertures and median aperture. Combined administration, or administration "prior to, simultaneous or after" intrathecal administration as used herein, means that administration into the cisterna magna and intrathecal administration take place within a time span of preferably less than 2 weeks, 1 week, 4 days, 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes or 5 minutes.

This step can be circumvented by the use of self-complementary vectors or monomeric duplex vectors which package an inverted repeat vector genome that folds into dsDNA without requiring DNA synthesis or base- pairing between multiple vector genomes, thereby increasing efficiency of AAV- mediated gene transfer. For a review of self- complementary AAV vectors, see e.

In a preferred embodiment, the self-complementary AAV vectors do not fall within the scope of the present invention. This has the advantage that multiple repeats concatemers of 2, 4, 8 or more times the expression unit can be built in the AAV vector resulting in mono, -duplo, - quadromeric duplex vectors. In the present invention it has been shown that broad transduction of brain tissue can be obtained after lumbar injection, using a single-stranded AAV gene therapy vector.

Therefore, it is a preferred embodiment of the invention that the AAV gene therapy vector is a single stranded AAV gene therapy vector. The AAV gene therapy vector of the present invention achieves transduction over a large area in the central nervous system after intrathecal lumbar administration. Using an ubiquitous promoter CAG promoter , both neuronal and glial cells were transduced in the cortex, cerebellum and subventricular zone. In addition, in the spinal cord, motorneuron transduction was prevalent as well as transduction of neurons in the dorsal root ganglia.

Hence, the method of treatment of the present invention can be used in the treatment of several CNS disorders. In a preferred embodiment, an AAV gene therapy vector according to the present invention comprises a gene product for interest that can be used in the treatment or prophylaxis of a condition selected from the group consisting of: amyotrofic laterale sclerose ALS , spinal muscular atrophy SMA , pain, lysosomal storage diseases LSD , Huntington's disease, Alzheimer's disease, Tay-Says disease, Friedreich ataxia, ataxia telangietacsia, Spinocerebellar ataxia type 1, 2 and 3, Niemann-Pick disease A, B and C, Dopa-responsive dystonia, Fragile X syndrome, Krabbe disease, Glycogen storage disease type 2 Pompe , Primary lateral sclerosis, Pelizaeus-Merzbacher disease, X-linked adrenoleukodystrophy, Giant axonal neuropathy, Multiple system atrophy MSA , Proximal myotonic myopathy, Neuronal Ceroid Lipofuscinosis Batten disease and various forms of CNS cancer, such as for example, primary CNS lymphoma, metastatic or secondary brain tumors, primary spinal cord tumors.

CNS cancer include for example glioblastoma, astrocytoma, oligodendroglioma, ependymoma, meningioma, medulloblastoma, ganglioglioma, schwannoma, craniopharyngioma, chordoma, non-Hodgkin CNS lymphoma. A group of metabolic disorders known as lysosomal storage diseases LSD includes over forty genetic disorders, many of which involve genetic defects in various lysosomal hydrolases. Representative lysosomal storage diseases and the associated defective enzymes are listed in Table 1. In a particularly preferred embodiment, an AAV gene therapy vector according to the present invention comprises a gene product for interest that can be used in the treatment or prophylaxis of a condition selected from the group consisting of: amyotrofic laterale sclerose ALS , spinal muscular atrophy SMA , Huntington's disease, Multiple system atrophy MSA and Lysosomal storage diseases selected from the group consisting of: Fabry, Juvenile Batten Disease CNL3 , Gaucher types 1, 2, and 3, Hunter, Pompe, Sanfilippo A and Sanfilippo B.

Moreover, miRNA sequences may be used as well to downregulate expression of certain genes, such as for example in the treatment of Huntington's disease. Most of the diseases mentioned herein, except for Huntington's disease, can be treated inter alia by provision of a corrected version of the defective gene. Huntington's disease however requires the downregulation of a certain gene, i. Approaches for the development of RNAi-based Huntington Disease therapy include total HTT knockdown of both mutant and wild type gene by for example targeting exon 1.

In a preferred embodiment the gene product of interest is operably linked to expression control elements comprising a promoter that produces sufficient expression of the gene product of interest to obtain a therapeutic effect. The level of transgene expression in eukaryotic cells is largely determined by the transcriptional promoter within the transgene expression cassette. Promoters that show long-term activity and are tissue and even cell-specific are used in some embodiments.

Nonlimiting examples of promoters include, but are not limited to, the cytomegalovirus CMV promoter Kaplitt et al. To prolong expression, other regulatory elements may additionally be operably finked to the transgene, such as, e. Virol, 72, or the bovine growth hormone BGH polyadenylation site. Drinkut and coworkers applied a glial GFAP promoter to express GDNF and showed that that was more beneficial than expressing this from a neuron specific promoter Drinkut et al, , supra.

Alternatively or in addition to use of a specific promoter, regulated expression could be achieved using regulatable expression elements such as for example the Gene Switch system Maddalena et al. Preferably the gene therapy vector is administered by lumbar injection, possibly in combination with injection into the cisterna magna.

A therapeutically effective amount of an AAV gene therapy vector or pharmaceutical composition of the invention may be given to a patient in need thereof. Such compositions include the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition in an effective amount, sufficient to provide a desired therapeutic or prophylactic effect, and a pharmaceutically acceptable carrier or excipient. An "effective amount" includes a therapeutically effective amount or a prophylactically effective amount.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as elevation of symptoms of the disorder. A therapeutically effective amount of a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition to elicit a desired response in the individual.

Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effects of the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting various conditions.

A prophylactic dose may be used in subjects prior to or at an earlier stage of disease, and a prophylactically effective amount may be more or less than a therapeutically effective amount in some cases. In particular embodiments, a range for therapeutically or prophylactically effective amounts of an AAV gene therapy vector or pharmaceutical composition may. It is to be noted that dosage values may vary with the severity of the condition to be alleviated.

For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. Preferably, the volume for infusion into the CSF in a human child is less than about 20cc and in a human adult less than about 30 cc.

In non-human primates 6 cc can safely be delivered into a CSF total volume of 20 - 25 cc. In addition, the AAV gene therapy vector of the invention can be administered in a concentrated form by standard methods known in the art, including but not limited to density-gradient centrifugation eg CsCl, iodixanol , dialysis, ultracentrifugation, ion exchange IEX chromatography eg anion, cation , gel filtration, affinity chromatography, Tangential Flow Filtration TFF , Spin filter columns eg Centricon.

As used herein "pharmaceutically acceptable carrier" or "exipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the invention. Guidance on co-administration of additional therapeutics may for example be found in the Compendium of Pharmaceutical and Specialties CPS of the Canadian Pharmacists Association. Vulchanova et al therefore suggest the pretreatment with mannitol to enhance the penetration of AAV5 and AAV8 vectors to the spinal cord parenchyma and to the cell bodies of DRG neurons.

In particular, lumbar injection of AAV vector in mice at a dosage of about 4 x 10 12 vector genomes per kg requires intravenous pretreatment with mannitol prior to intrathecal lumbar administration of the vector in order to obtain expression of the reporter gene green fluorescent protein GFP in the spinal cord and select subsets of DRG neurons.

The pre-treatment with mannitol has as drawback that mannitol is known to cause side effects. In particular, adverse reactions more commonly reported during or after the infusion of mannitol include: Pulmonary congestion, fluid and electrolyte imbalance, acidosis, electrolyte loss, dryness of mouth, thirst, marked diuresis, urinary retention, edema, headache, blurred vision, convulsions, nausea, vomiting, rhinitis, arm pain, skin necrosis, thrombophlebitis, chills, dizziness, urticaria, dehydration, hypotension, tachycardia, fever and angina-like chest pains Mannitol IV FDA Prescribing Information.

In a preferred embodiment of the present invention, the subject is not subjected to pretreatment prior to administration of the AAV gene therapy vector. More preferably, the subject is not subjected to pretreatment with an osmotic agent prior to administration of the AAV gene therapy vectors. Osmotic agents include, but are not limited to urea, glycerol, sugars or sugar- alcohols such as polyols like mannitol and hypertonic saline. In particular, the subject is not subjected to pretreatment with mannitol, such as intravenous mannitol pretreatment prior to intrathecal lumbar administration of the AAV gene therapy vector.

In a preferred embodiment of the present invention, the mammalian subject includes but is not limited to murines, rats, simians, farm animals, sport animals, pets and human. More preferably, the mammalian subject is a human. In an additional aspect, the disclosure relates to a method for producing a recombinant parvoviral for example rAAV virion comprising a recombinant parvoviral rAAV vector as defined above in an insect cell.

Preferably, the method comprises the steps of: a culturing an insect cell as defined herein under conditions such that recombinant parvoviral for example rAAV vector is produced; and, b recovery of the recombinant parvoviral for example rAAV vector. It is understood here that the recombinant parvoviral rAAV vector produced in the method preferably is an infectious parvoviral or AAV virion that comprise the recombinant parvoviral rAAV vector nucleic acids.

Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art and described e. Preferred methods and constructs for the production of rAAV virions of the invention are disclosed in e. Preferably the method for producing recombinant parvoviral virions further comprises the step of affinity-purification of the virions comprising the recombinant parvoviral rAAV vector using an anti-AAV antibody, preferably an immobilised antibody.

The anti-AAV antibody preferably is an monoclonal antibody. A particularly suitable antibody is a single chain camelid antibody or a fragment thereof as e. Muyldermans, , Biotechnol. The antibody for affinity-purification of rAAV preferably is an antibody that specifically binds an epitope on an AAV capsid protein, whereby preferably the epitope is an epitope that is present on capsid proteins of more than one AAV serotype. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

Gene Therapy

This volume aims to explore the latest developments in adeno-associated viral and lentiviral vectors as well as the gene therapy strategies for the most common . Gene therapy approaches to diseases of the nervous system are very of the currently available gene therapy methods, including gene delivery vehicles.

In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Figure 1. The expression cassette encoding for a GOI gene of interest or gene product of interest , is under control of the CAG promoter, preceded by a Kozak sequence gccaccatg This cassette is flanked by two non-coding ITR sequences.

Using recombinant baculoviruses for production, the expression cassete is packaged into an AAV-5 capsid. Figure 2. Overview of transduction of the brain. Expression is observed throughout the brain. Figure 3. Higher magnification of the parietal cortex. Both neuronal and glial transduction can be observed several layers deep. Figure 4. Higher magnification of the occipital cortex. Figure 5. Higher magnification of the cerebellum. Many cells are positive for GFP. Figure 6. Transduction of the cervical spinal cord. Sensory fibers terminating in the grey matter as well as ascending fibers as dots can be seen a.

Moreover, GFP positive motorneuron fibers b coming from the motorneuron pool can be observed c. In d some glial cell are also stained positive for GFP. Figure 7. Transduction of the thoracic spinal cord. Ascending sensory fibers fibers can be seen in the dorsal column a. In d some fibers are observed that can be both descending or ascending.

Figure 8. Transduction of the lumbar spinal cord. Sensory fibers fibers terimianting in the grey matter are visible a. Moreover, GFP positive motorneurons in the motorneuronpool are present in the dorsolateral part of the spinal cord. These motorneurons project their fibers into the ventral root through the ventral part of the spinal cord d. Figure 9. Transduction of the Dorsal root ganglia along the spinal cord can be seen. Almost all DRG neurons are transduced. Figure The arrowhead is pointing at a neuron that is expressing GFP in the cortex.

The arrow is pointing at an astrocyte expressing GFP. The arrowhead is pointing at a cell that has characteristics of a neuron. Haematoxilin Eosin staining of sections of the brain and cerebellum. Top left is an overview of the cerebellum. Top right is a higher magnification of the molecular layer of the cerebellum.

Nicely aligned are the large round neurons see also inset , the Purkinje cells. In general, no obvious infiltrates or irregularities are observed indicating that the treatment is well tolerated. GFP stained sections of the lumbar spinal cord showing dose dependent transduction of motorneurons in the lumbar spinal cord by presence of the transgene of large neurons in the ventral horn.

Higher magnifications are shown of the ventral horn, where the motorneurons are located, are depicted B, D and F. Transgene expression was examined at 4 weeks post-injection. Moreover, in the cerebellum many Bergman glia were transduced as well as some Purkinje cells. In the brain, the cortex was also expressing GFP in both neurons and glia. Cells along the subventricular zone SVZ were also transduced. Altogether, these results indicate that using this route of injection, a large area of the CNS is covered and gene therapy for the CNS becomes a realistic option.

Expression is under the control of The CAG promoter, a combination of the cytomegalovirus CMV early enhancer element and chicken beta-actin promoter. The whole cassette is flanked by two non-coding inverted terminal repeats of AAV-2 see Figure 1. Recombinant AAV-5 vectors were prepared using a baculovirus expression system similar as described earlier Urabe et al, , Unzu et al, , reviewed in Kotin, Briefly, three recombinant baculoviruses, one encoding for the REP for replication and packaging, one encoding for the CAP-5 for the capsid of AAV-5 and one with the expression cassette, were used to infect SF9 insect cells.

Titer of the vector was 1. Daily health observations and periodic body weight measurements were recorded in order to assess the welfare of the animals. Veterinarians reported no abnormal symptoms throughout the study. Before starting the procedure, animals were negatively tested for the presence of neutralizing antibodies against AAV see below.

After surgery, all animals received an intramuscular dose of atipamezole hydrochloride to reverse anesthesia Antisedan, 0. Brains and organs were collected and processed for histological analysis. Sections were then placed in cryoprotectant solution until use. After induction of anesthesia, the animal's head was placed in a stereotactic frame and flexed in a prone position, and the back of the neck was cleaned with povidone-iodine and alcohol. A 3 -ml syringe attached to a 1-inch 23 -gauge needle was mounted onto the micromanipulator, and the needle was manually guided into the Cisterna magna.

The lumbar sac was approached similarly between the spinal level of L4 and 5. Penetration was verified by aspiration of a small volume of cerebrospinal fluid CSF into the syringe, which was then secured to the micromanipulator. The three-way stopcock on the syringe was then adjusted to allow infusion of 3 ml of vector at 0. After completion of the infusion, the line was flushed with 0. After verification, the needle was slowly removed. Hence, each animal received 3 ml into the cisterna magna and 3 ml by lumbar injection.

In total, 6 ml of 1.

Therapeutic nanoplatforms and delivery strategies for neurological disorders

For a 5kg monkey, this constitutes 2. Therefore, the administrated dose of 5. Since lumbar infusion of a blue dye results in blue dye that is tapped cisternally, no differences in outcome are expected if the gene therapy vector is only administered lumbar. Chromogenic and immunofluorescence staining were performed on lm coronal sections throughout four alternate 6-mm blocks from the prefrontal cortex to the cerebellum and the spinal cord. Chromogenic GFP staining was performed as described previously Hadaczek et al. Sections were then mounted on slides, dehydrated, and coverslipped with Shandon-Mount cat.

No ; Thermo Fisher Scientific. To determine the phenotype of GFP-positive cells, double immunofluorescence staining was performed. Sections were coverslipped with a 4 ,6-diamidino- 2-phenylindole DAPI - containing hard-set media to identify all nuclei Vectashield H; Vector Laboratories. Sera were diluted over a range from 1 to 1 and incubated at room temperature for 1 hr. Louis, MO at room temperature for 1 hr. The reaction was stopped by the addition of hydrochloric acid and absorbance was read at nm on a plate reader Bevan et al.

Pre-Dose: Blood samples were collected from adult cynomolgus macaques Macaca fasciularis for screening and analyzed for serum anti-AAV-5 antibodies titer, serum clinical chemistry and hematology evaluations. Tissue harvesting: The CSF-infused animals were euthanized at 8 weeks post-infusion. The animals were dosed with euthanasia solution. Following verification of a deep plane of anesthesia i.

The brain and spinal cord were harvested and a tissue punch was taken for molecular Q-PCR analysis. The remainder of the tissue was saved in paraformaldehyde for histological evaluations. The outcome is depicted in table 2. The procedure was well tolerated by the animals. No weight loss was observed and also no clinical signs were noted by the veterinarian. As indicated in figure 14, administration of a regular AAV5 dose 5E13 GC resulted in hardly any transduction, confirming the consensus in the art that AAV5 only poorly transduces neuronal cells following intrathecal administration.

Surprisingly, higher doses of AAV5 resulted in a significant transduction of motorneurons. Figure 14 shows that there is an increasing transduction of motorneurons when a higher dose is used. In the dorsal horn, some fibers are also observed, suggesting transduction of DRG neurons. However, the predominant transduced cell type constitute motor neurons.

Furthermore, the presence of AAV genomes was determined in the spinal cord, brain and liver. For example, Haurigot et al , J. Of Clin. Interestingly, in contrast to e. The liver is not invaded by AAV5 Table 2. The following observations derive from experiments performed with 8. Expression was observed in both neuronal and glial cells.

In particular, GFP expression was predominantly observed in the motor neurons. Vulchanova et al also observe transduction of neuronal cells with AAV5, after pretreatment with mannitol. However, Vulchanova et al see a much higher transduction efficiency of DRG neurons and their neurites projecting into the dorsal horn of the spinal cord in comparison the motor neurons. An overview several slices of the brain showed nice expression throughout the whole brain, mainly in relative close vicinity of the ventricle walls see Figure 2.

Expression in the parietal cortex showed expression through several layers of the cortex. Also some astrocytes were transduced Figure 3. The same holds true for the occipital part of the cortex Figure 4. In the cerebellum, the windings of the structure of the cerebellum were clearly depicted by the line of transduced cells Figure 5.

Also in this part of the brain, both neuronal and glial cell transduction is observed. In the spinal cord, many if not all motorneurons were transduced over the entire length of the spinal cord Figure 6, 7 and 8. A similar observation was made for the neurons in the DRG Figure 9. They were also heavily transduced to express GFP. To identify the cells that were expressing the transgene, double staining for neuronal and astrocytic markers was performed. Using this specific CAG promoter, bot neuronal and astrocytic transduction was achieved Figures 10, 11 and Based on the remaining presence of the Purkinje cell layer, no neurotoxicity was observed using this specific approach.

Following a single injection into the brain, expression of the transgene is local and remains mainly restricted to the infused area. The treatment was well tolerated as no clinical signs and no obvious neuronal loss were observed. Both neuronal and glial cells were transduced using this specific CAG promoter.

Using cell specific promoters, such as the GFAP promoter or synapsin-1 promoter, one could direct the expression to a specific population. We have shown that areas of the cortex, cerebellum and sub ventricular zone show expression of the transgene in both neurons and glial cells. Indications that could be helped by this could be: Motorneuron diseases such as amyotrofic laterale sclerose ALS , spinal muscular atrophy SMA , or sensory related indications such as pain. Other neurological indications that could be helped by such an approach are for instance Huntington's disease, Alzheimer's disease, Tay-Says disease, Friedreich ataxia, ataxia telangietacsia, Spinocerebellar ataxia type 1, 2 and 3 , Niemann-Pick disease A, B and C , Dopa-responsive dystonia, Fragile X syndrome, Krabbe disease, Glycogen storage disease type 2 Pompe , Primary lateral sclerosis, Pelizaeus-Merzbacher disease, X-linked adrenoleukodystrophy, Giant axonal neuropathy, Multiple system atrophy MSA , Proximal myotonic myopathy, and Neuronal Ceroid Lipofuscinosis Batten disease.

The genetic defects that are responsible are largely known and could be built into an AAV -5 vector. Using this delivery system as a therapeutic agent, one could imagine that this reverses the genotype and hence hopefully also the phenotype. Moreover, the intrathecal method of delivery is regarded as less invasive as infusion into the CNS itself as brain surgery requires complex surgery.

The intrathecal approach has as additional value that this could be performed without the need of a specialized center for brain injections. J Virol. Human adeno-associated virus type 5 is only distantly related to other known primate helper-dependent parvoviruses. Hum Gene Ther. Selective and rapid uptake of adeno-associated virus type 2 in brain. Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders. Mol Ther. J Neurosci Methods. Adeno-associated viral vector AAV -mediated gene transfer in the red nucleus of the adult rat brain: comparative analysis of the transduction properties of seven AAV serotypes and lentiviral vectors.

Gene Therapy for Neurological Disorders | Taylor & Francis Group

As may be deduced from the name of their genus, members of the Dependovirus are unique in that they usually require co-infection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture. The genus Dependovirus includes AAV, which normally infects humans e. Further information on parvoviruses and other members of the Parvoviridae is described in Kenneth I. The genomic organization of all known AAV serotypes is very similar.

Inverted terminal repeats ITRs flank the unique coding nucleotide sequences for the non-structural replication Rep proteins and the structural VP proteins.

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The VP proteins VPl, -2 and -3 form the capsid. The terminal nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild-type wt AAV infection in mammalian cells the Rep genes i. Rep78 and Rep52 are expressed from the P5 promoter and the P19 promoter, respectively and both Rep proteins have a function in the replication of the viral genome. Rep78, Rep68, Rep52 and Rep A "recombinant parvoviral or AAV vector" or "rAAV vector" herein refers to a vector comprising one or more polynucleotide sequences of interest, a gene product of interest, genes of interest or "transgenes" that are flanked by at least one parvoviral or AAV inverted terminal repeat sequences ITRs.

Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that is expressing AAV rep and cap gene products i. AAV Rep and Cap proteins. When an rAAV vector is incorporated into a larger nucleic acid construct e. AAV is able to infect a number of mammalian cells. See, e. Cell Biol. Gene Ther. However, AAV transduction of human synovial fibroblasts is significantly more efficient than in similar murine cells, Jennings et al, Arthritis Res, 3 : 1 , and the cellular tropicity of AAV differs among serotypes.

An AAV gene therapy vector for use in the present invention may be produced either in mammalian cells or in insect cells. Both methods are described in the art. For example Grimm et al. This reference is herein included in its entirety. Further information can also be found in Blits et al. The term "chimeric" is used herein to describe that a single gene, such as for example the capsid, is composed of at least two sequences derived from different serotypes.

AAV5 can for example be produced in mammalian cells according to the following method, but is not limited thereto: The vector genome contains the transgene expression cassette flanked by two inverted terminal repeats ITRs derived from AAV serotype 2. The total length of the viral vector genome may not exceed the wild type genome size of 4. The manufacturing process of AAV vectors is based upon Ca P04 2 transfection of two plasmids into human embryonic kidney production cells HEK in roller bottles cm 2 surface area followed by purification of the encapsidated vector genomes by filtration and chromatography techniques.

The genome of the production cell line comprises the adenovirus El to provide helper functions. Vector production in roller bottles on average results in yields of 3x10 3 vector genomes per cell or 4x10 11 vector genomes per roller bottle quantified by qPCR. Subsequently, the cell culture is lysed by a buffer containing Triton-X and cell debris removed by low speed centrifugation.

Alternatively, an AAV gene therapy vector for use in the present invention may be produced in insect cells, as has been described previously by Urabe et al.

Whole-genome Testing and the Possibility of Gene Therapy for Neurological Disorders

Journal of Virology 80 4 : Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels. This provides an identical set of genetic functions to produce virions which are essentially physically and functionally equivalent. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e. The Rep78 proteins of various AAV serotypes are e.

Virol, 73 2 The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid proteins of one serotype e. Such pseudotyped rAAV particles are a part of the present invention. Modified "AAV" sequences also can be used in the context of the present invention, e. Such modified sequences e.

In view thereof, the production of rAAV5 can differ from production of other serotypes in insect cells. Where methods of the invention are employed to produce rAAV5, it is preferred that one or more constructs comprise, collectively in the case of more than one construct, a nucleotide sequence comprising an AAV5 ITR, a nucleotide sequence comprises an AAV5 Rep coding sequence i. Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell , J.

Data Standards Manual. Food and Drug Administration. Retrieved 11 March This is an injection through the theca of the spinal cord into the subarachnoid space so that it reaches the CSF. This method of administration is useful in for example spinal anaesthesia, chemotherapy, or pain management applications. This route is also used to introduce drugs that fight certain infections, particularly post-neurosurgical.

A substance administered via intrathecal injection avoids the need to passage the blood brain barrier. Drugs given intrathecally often do not contain any preservative or other potentially harmful inactive ingredients that are sometimes found in medicaments for intravenous injection. The intrathecal method of delivery is regarded as less invasive than infusion into the central nervous system CNS tissue itself, because infusion into the CNS requires complex brain surgery. Intrathecal delivery can be performed without the need of a specialized center for brain injections.

The neck region of the spine is known as the Cervical Spine. This region consists of seven vertebrae, which are abbreviated CI through C7 top to bottom. These vertebrae protect the brain stem and the spinal cord, support the skull, and allow for a wide range of head movement. The first cervical vertebra CI is called the Atlas.

The Atlas is ring-shaped and it supports the skull. C2 is called the Axis. It is circular in shape with a blunt tooth-like structure called the Odontoid Process or dens that projects upward into the Atlas. Together, the Atlas and Axis enable the head to rotate and turn. The other cervical vertebrae C3 through C7 are shaped like boxes with small spinous processes finger-like projections that extend from the back of the vertebrae.

Beneath the last cervical vertebra are the 12 vertebrae of the Thoracic Spine. These are abbreviated Tl through T12 top to bottom. Tl is the smallest and T12 is the largest thoracic vertebra. The thoracic vertebrae are larger than the cervical bones and have longer spinous processes. In addition to longer spinous processes, rib attachments add to the thoracic spine's strength.

These structures make the thoracic spine more stable than the cervical or lumbar regions. In addition, the rib cage and ligament system limit the thoracic spine's range of motion and protects many vital organs. The size and shape of each lumbar vertebra is designed to carry most of the body's weight. Each structural element of a lumbar vertebra is bigger, wider and broader than similar components in the cervical and thoracic regions. The lumbar spine has more range of motion than the thoracic spine, but less than the cervical spine.

The lumbar facet joints allow for significant flexion and extension movement but limits rotation. The Sacrum is located behind the pelvis. Five bones, abbreviated SI through S5, fused into a triangular shape, form the sacrum. The sacrum fits between the two hip bones connecting the spine to the pelvis. The last lumbar vertebra L5 articulates moves with the sacrum. Immediately below the sacrum are five additional bones, fused together to form the Coccyx tailbone.

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In a preferred embodiment of the present invention, the gene therapy vector is administered by lumbar administration. Most preferably, the gene therapy vector is administered by intrathecal injection between L4 and L5. In one preferred embodiment, the gene therapy vector is administrated only by lumbar administration, i. Alternatively or in combination with a previously preferred embodiment of the present invention, in a preferred embodiment of the invention the gene therapy vector is administered into the cisterna magna.

In one preferred embodiment, the gene therapy vector is administrated only into the cisterna magna, i. More preferably, administration into the cisterna magna is performed in combination with the intrathecal administration, or in other words prior to, simultaneous or after the intrathecal administration.

In another preferred embodiment, administration into the cisterna magna is performed in combination with the lumbar administration, whereby preferably administration into the cisterna magna is performed prior to, simultaneous or after the lumbar administration. Preferably in this embodiment, the lumbar administration is in the intrathecal region.

In a most preferred embodiment, the gene therapy vector is administered only into the cisterna magna in combination with the lumbar administration, i. The "cisterna magna" or "cerebellomedullary cistern" is one of three principal openings in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain. The openings are collectively referred to as cisterns. The cisterna magna is located between the cerebellum and the dorsal surface of the medulla oblongata. Cerebrospinal fluid produced in the fourth ventricle drains into the cisterna magna via the lateral apertures and median aperture.

Combined administration, or administration "prior to, simultaneous or after" intrathecal administration as used herein, means that administration into the cisterna magna and intrathecal administration take place within a time span of preferably less than 2 weeks, 1 week, 4 days, 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes or 5 minutes. This step can be circumvented by the use of self-complementary vectors or monomeric duplex vectors which package an inverted repeat vector genome that folds into dsDNA without requiring DNA synthesis or base- pairing between multiple vector genomes, thereby increasing efficiency of AAV- mediated gene transfer.

For a review of self- complementary AAV vectors, see e. In a preferred embodiment, the self-complementary AAV vectors do not fall within the scope of the present invention. This has the advantage that multiple repeats concatemers of 2, 4, 8 or more times the expression unit can be built in the AAV vector resulting in mono, -duplo, - quadromeric duplex vectors. In the present invention it has been shown that broad transduction of brain tissue can be obtained after lumbar injection, using a single-stranded AAV gene therapy vector. Therefore, it is a preferred embodiment of the invention that the AAV gene therapy vector is a single stranded AAV gene therapy vector.

The AAV gene therapy vector of the present invention achieves transduction over a large area in the central nervous system after intrathecal lumbar administration. Using an ubiquitous promoter CAG promoter , both neuronal and glial cells were transduced in the cortex, cerebellum and subventricular zone. In addition, in the spinal cord, motorneuron transduction was prevalent as well as transduction of neurons in the dorsal root ganglia. Hence, the method of treatment of the present invention can be used in the treatment of several CNS disorders.

In a preferred embodiment, an AAV gene therapy vector according to the present invention comprises a gene product for interest that can be used in the treatment or prophylaxis of a condition selected from the group consisting of: amyotrofic laterale sclerose ALS , spinal muscular atrophy SMA , pain, lysosomal storage diseases LSD , Huntington's disease, Alzheimer's disease, Tay-Says disease, Friedreich ataxia, ataxia telangietacsia, Spinocerebellar ataxia type 1, 2 and 3, Niemann-Pick disease A, B and C, Dopa-responsive dystonia, Fragile X syndrome, Krabbe disease, Glycogen storage disease type 2 Pompe , Primary lateral sclerosis, Pelizaeus-Merzbacher disease, X-linked adrenoleukodystrophy, Giant axonal neuropathy, Multiple system atrophy MSA , Proximal myotonic myopathy, Neuronal Ceroid Lipofuscinosis Batten disease and various forms of CNS cancer, such as for example, primary CNS lymphoma, metastatic or secondary brain tumors, primary spinal cord tumors.

CNS cancer include for example glioblastoma, astrocytoma, oligodendroglioma, ependymoma, meningioma, medulloblastoma, ganglioglioma, schwannoma, craniopharyngioma, chordoma, non-Hodgkin CNS lymphoma. A group of metabolic disorders known as lysosomal storage diseases LSD includes over forty genetic disorders, many of which involve genetic defects in various lysosomal hydrolases.

Representative lysosomal storage diseases and the associated defective enzymes are listed in Table 1. In a particularly preferred embodiment, an AAV gene therapy vector according to the present invention comprises a gene product for interest that can be used in the treatment or prophylaxis of a condition selected from the group consisting of: amyotrofic laterale sclerose ALS , spinal muscular atrophy SMA , Huntington's disease, Multiple system atrophy MSA and Lysosomal storage diseases selected from the group consisting of: Fabry, Juvenile Batten Disease CNL3 , Gaucher types 1, 2, and 3, Hunter, Pompe, Sanfilippo A and Sanfilippo B.

Moreover, miRNA sequences may be used as well to downregulate expression of certain genes, such as for example in the treatment of Huntington's disease.

Most of the diseases mentioned herein, except for Huntington's disease, can be treated inter alia by provision of a corrected version of the defective gene. Huntington's disease however requires the downregulation of a certain gene, i. Approaches for the development of RNAi-based Huntington Disease therapy include total HTT knockdown of both mutant and wild type gene by for example targeting exon 1.

In a preferred embodiment the gene product of interest is operably linked to expression control elements comprising a promoter that produces sufficient expression of the gene product of interest to obtain a therapeutic effect. The level of transgene expression in eukaryotic cells is largely determined by the transcriptional promoter within the transgene expression cassette. Promoters that show long-term activity and are tissue and even cell-specific are used in some embodiments. Nonlimiting examples of promoters include, but are not limited to, the cytomegalovirus CMV promoter Kaplitt et al.

To prolong expression, other regulatory elements may additionally be operably finked to the transgene, such as, e. Virol, 72, or the bovine growth hormone BGH polyadenylation site. Drinkut and coworkers applied a glial GFAP promoter to express GDNF and showed that that was more beneficial than expressing this from a neuron specific promoter Drinkut et al, , supra.

Alternatively or in addition to use of a specific promoter, regulated expression could be achieved using regulatable expression elements such as for example the Gene Switch system Maddalena et al. Preferably the gene therapy vector is administered by lumbar injection, possibly in combination with injection into the cisterna magna.

A therapeutically effective amount of an AAV gene therapy vector or pharmaceutical composition of the invention may be given to a patient in need thereof. Such compositions include the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition in an effective amount, sufficient to provide a desired therapeutic or prophylactic effect, and a pharmaceutically acceptable carrier or excipient. An "effective amount" includes a therapeutically effective amount or a prophylactically effective amount.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as elevation of symptoms of the disorder. A therapeutically effective amount of a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition to elicit a desired response in the individual.

Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effects of the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting various conditions. A prophylactic dose may be used in subjects prior to or at an earlier stage of disease, and a prophylactically effective amount may be more or less than a therapeutically effective amount in some cases.

In particular embodiments, a range for therapeutically or prophylactically effective amounts of an AAV gene therapy vector or pharmaceutical composition may. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.

Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. Preferably, the volume for infusion into the CSF in a human child is less than about 20cc and in a human adult less than about 30 cc. In non-human primates 6 cc can safely be delivered into a CSF total volume of 20 - 25 cc. In addition, the AAV gene therapy vector of the invention can be administered in a concentrated form by standard methods known in the art, including but not limited to density-gradient centrifugation eg CsCl, iodixanol , dialysis, ultracentrifugation, ion exchange IEX chromatography eg anion, cation , gel filtration, affinity chromatography, Tangential Flow Filtration TFF , Spin filter columns eg Centricon.

As used herein "pharmaceutically acceptable carrier" or "exipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the invention. Guidance on co-administration of additional therapeutics may for example be found in the Compendium of Pharmaceutical and Specialties CPS of the Canadian Pharmacists Association. Vulchanova et al therefore suggest the pretreatment with mannitol to enhance the penetration of AAV5 and AAV8 vectors to the spinal cord parenchyma and to the cell bodies of DRG neurons. In particular, lumbar injection of AAV vector in mice at a dosage of about 4 x 10 12 vector genomes per kg requires intravenous pretreatment with mannitol prior to intrathecal lumbar administration of the vector in order to obtain expression of the reporter gene green fluorescent protein GFP in the spinal cord and select subsets of DRG neurons.

The pre-treatment with mannitol has as drawback that mannitol is known to cause side effects. In particular, adverse reactions more commonly reported during or after the infusion of mannitol include: Pulmonary congestion, fluid and electrolyte imbalance, acidosis, electrolyte loss, dryness of mouth, thirst, marked diuresis, urinary retention, edema, headache, blurred vision, convulsions, nausea, vomiting, rhinitis, arm pain, skin necrosis, thrombophlebitis, chills, dizziness, urticaria, dehydration, hypotension, tachycardia, fever and angina-like chest pains Mannitol IV FDA Prescribing Information.

In a preferred embodiment of the present invention, the subject is not subjected to pretreatment prior to administration of the AAV gene therapy vector. More preferably, the subject is not subjected to pretreatment with an osmotic agent prior to administration of the AAV gene therapy vectors. Osmotic agents include, but are not limited to urea, glycerol, sugars or sugar- alcohols such as polyols like mannitol and hypertonic saline.

In particular, the subject is not subjected to pretreatment with mannitol, such as intravenous mannitol pretreatment prior to intrathecal lumbar administration of the AAV gene therapy vector. In a preferred embodiment of the present invention, the mammalian subject includes but is not limited to murines, rats, simians, farm animals, sport animals, pets and human. More preferably, the mammalian subject is a human. In an additional aspect, the disclosure relates to a method for producing a recombinant parvoviral for example rAAV virion comprising a recombinant parvoviral rAAV vector as defined above in an insect cell.

Preferably, the method comprises the steps of: a culturing an insect cell as defined herein under conditions such that recombinant parvoviral for example rAAV vector is produced; and, b recovery of the recombinant parvoviral for example rAAV vector. It is understood here that the recombinant parvoviral rAAV vector produced in the method preferably is an infectious parvoviral or AAV virion that comprise the recombinant parvoviral rAAV vector nucleic acids. Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art and described e.

Our Approach & Programs

Preferred methods and constructs for the production of rAAV virions of the invention are disclosed in e. Preferably the method for producing recombinant parvoviral virions further comprises the step of affinity-purification of the virions comprising the recombinant parvoviral rAAV vector using an anti-AAV antibody, preferably an immobilised antibody. The anti-AAV antibody preferably is an monoclonal antibody. A particularly suitable antibody is a single chain camelid antibody or a fragment thereof as e. Muyldermans, , Biotechnol. The antibody for affinity-purification of rAAV preferably is an antibody that specifically binds an epitope on an AAV capsid protein, whereby preferably the epitope is an epitope that is present on capsid proteins of more than one AAV serotype.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Figure 1. The expression cassette encoding for a GOI gene of interest or gene product of interest , is under control of the CAG promoter, preceded by a Kozak sequence gccaccatg This cassette is flanked by two non-coding ITR sequences.

Using recombinant baculoviruses for production, the expression cassete is packaged into an AAV-5 capsid. Figure 2. Overview of transduction of the brain. Expression is observed throughout the brain. Figure 3. Higher magnification of the parietal cortex.

Both neuronal and glial transduction can be observed several layers deep. Figure 4. Higher magnification of the occipital cortex. Figure 5. Higher magnification of the cerebellum. Many cells are positive for GFP. Figure 6. Transduction of the cervical spinal cord. Sensory fibers terminating in the grey matter as well as ascending fibers as dots can be seen a.

Moreover, GFP positive motorneuron fibers b coming from the motorneuron pool can be observed c. In d some glial cell are also stained positive for GFP. Figure 7. Transduction of the thoracic spinal cord. Ascending sensory fibers fibers can be seen in the dorsal column a. In d some fibers are observed that can be both descending or ascending. Figure 8. Transduction of the lumbar spinal cord. Sensory fibers fibers terimianting in the grey matter are visible a. Moreover, GFP positive motorneurons in the motorneuronpool are present in the dorsolateral part of the spinal cord. These motorneurons project their fibers into the ventral root through the ventral part of the spinal cord d.

Figure 9. Transduction of the Dorsal root ganglia along the spinal cord can be seen. Almost all DRG neurons are transduced. Figure The arrowhead is pointing at a neuron that is expressing GFP in the cortex. The arrow is pointing at an astrocyte expressing GFP. The arrowhead is pointing at a cell that has characteristics of a neuron.

Haematoxilin Eosin staining of sections of the brain and cerebellum. Top left is an overview of the cerebellum. Top right is a higher magnification of the molecular layer of the cerebellum. Nicely aligned are the large round neurons see also inset , the Purkinje cells. In general, no obvious infiltrates or irregularities are observed indicating that the treatment is well tolerated. GFP stained sections of the lumbar spinal cord showing dose dependent transduction of motorneurons in the lumbar spinal cord by presence of the transgene of large neurons in the ventral horn.

Higher magnifications are shown of the ventral horn, where the motorneurons are located, are depicted B, D and F. Transgene expression was examined at 4 weeks post-injection. Moreover, in the cerebellum many Bergman glia were transduced as well as some Purkinje cells. In the brain, the cortex was also expressing GFP in both neurons and glia. Cells along the subventricular zone SVZ were also transduced. Altogether, these results indicate that using this route of injection, a large area of the CNS is covered and gene therapy for the CNS becomes a realistic option.

Expression is under the control of The CAG promoter, a combination of the cytomegalovirus CMV early enhancer element and chicken beta-actin promoter. The whole cassette is flanked by two non-coding inverted terminal repeats of AAV-2 see Figure 1. Recombinant AAV-5 vectors were prepared using a baculovirus expression system similar as described earlier Urabe et al, , Unzu et al, , reviewed in Kotin, Briefly, three recombinant baculoviruses, one encoding for the REP for replication and packaging, one encoding for the CAP-5 for the capsid of AAV-5 and one with the expression cassette, were used to infect SF9 insect cells.

Titer of the vector was 1. Daily health observations and periodic body weight measurements were recorded in order to assess the welfare of the animals. Veterinarians reported no abnormal symptoms throughout the study. Before starting the procedure, animals were negatively tested for the presence of neutralizing antibodies against AAV see below. After surgery, all animals received an intramuscular dose of atipamezole hydrochloride to reverse anesthesia Antisedan, 0. Brains and organs were collected and processed for histological analysis.

Sections were then placed in cryoprotectant solution until use. After induction of anesthesia, the animal's head was placed in a stereotactic frame and flexed in a prone position, and the back of the neck was cleaned with povidone-iodine and alcohol. A 3 -ml syringe attached to a 1-inch 23 -gauge needle was mounted onto the micromanipulator, and the needle was manually guided into the Cisterna magna. The lumbar sac was approached similarly between the spinal level of L4 and 5.

Penetration was verified by aspiration of a small volume of cerebrospinal fluid CSF into the syringe, which was then secured to the micromanipulator. The three-way stopcock on the syringe was then adjusted to allow infusion of 3 ml of vector at 0. After completion of the infusion, the line was flushed with 0. After verification, the needle was slowly removed. Hence, each animal received 3 ml into the cisterna magna and 3 ml by lumbar injection. In total, 6 ml of 1. For a 5kg monkey, this constitutes 2. Therefore, the administrated dose of 5. Since lumbar infusion of a blue dye results in blue dye that is tapped cisternally, no differences in outcome are expected if the gene therapy vector is only administered lumbar.

Chromogenic and immunofluorescence staining were performed on lm coronal sections throughout four alternate 6-mm blocks from the prefrontal cortex to the cerebellum and the spinal cord. Chromogenic GFP staining was performed as described previously Hadaczek et al.

Sections were then mounted on slides, dehydrated, and coverslipped with Shandon-Mount cat. No ; Thermo Fisher Scientific. To determine the phenotype of GFP-positive cells, double immunofluorescence staining was performed. Sections were coverslipped with a 4 ,6-diamidino- 2-phenylindole DAPI - containing hard-set media to identify all nuclei Vectashield H; Vector Laboratories. Sera were diluted over a range from 1 to 1 and incubated at room temperature for 1 hr.

Louis, MO at room temperature for 1 hr. The reaction was stopped by the addition of hydrochloric acid and absorbance was read at nm on a plate reader Bevan et al. Pre-Dose: Blood samples were collected from adult cynomolgus macaques Macaca fasciularis for screening and analyzed for serum anti-AAV-5 antibodies titer, serum clinical chemistry and hematology evaluations. Tissue harvesting: The CSF-infused animals were euthanized at 8 weeks post-infusion.

The animals were dosed with euthanasia solution. Following verification of a deep plane of anesthesia i. The brain and spinal cord were harvested and a tissue punch was taken for molecular Q-PCR analysis. The remainder of the tissue was saved in paraformaldehyde for histological evaluations. The outcome is depicted in table 2. The procedure was well tolerated by the animals. No weight loss was observed and also no clinical signs were noted by the veterinarian. As indicated in figure 14, administration of a regular AAV5 dose 5E13 GC resulted in hardly any transduction, confirming the consensus in the art that AAV5 only poorly transduces neuronal cells following intrathecal administration.

Surprisingly, higher doses of AAV5 resulted in a significant transduction of motorneurons. Figure 14 shows that there is an increasing transduction of motorneurons when a higher dose is used. In the dorsal horn, some fibers are also observed, suggesting transduction of DRG neurons. However, the predominant transduced cell type constitute motor neurons. Furthermore, the presence of AAV genomes was determined in the spinal cord, brain and liver.

For example, Haurigot et al , J. Of Clin. Interestingly, in contrast to e. The liver is not invaded by AAV5 Table 2.