Gene Delivery and Therapy for Neurological Disorders

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The protein leads to the production of so-called regulatory T cells, which suppress the rogue immune system cells responsible for attacking the protective layer of nerve cells that defines multiple sclerosis. The effectiveness of this approach is based on targeting the gene therapy to the liver, which promotes immune tolerance. Hoffman, Ph.

Ben Philpot: Gene Awakenings for the Treatment of Neurological Disorders

The gene therapy-induced protein, MOG, was found to be effective in preventing and reversing multiple sclerosis on its own and the results have been validated through multiple independent experiments. Overall, the therapy was tested in groups of five to 10 mice and the results were reproduced multiple times. Hoffman said he was also encouraged by the treatment's longevity. After seven months, the mouse models that were treated with gene therapy showed no signs of disease, compared with a control group that had neurological problems after 14 days.

When the gene therapy was combined with immunotherapy using rapamycin -- a drug used to coat heart stents and prevent organ transplant rejection -- its effectiveness was further improved, the researchers found. The drug was chosen because it allows helpful regulatory T-cells to proliferate while blocking undesirable effector T-cells, Hoffman said. Among the two groups that were given rapamycin and the gene therapy, 71 percent and 80 percent went into near-complete remission after having hind-limb paralysis or near quadriplegia, respectively.

That, Hoffman said, shows how powerful and effective the combination can be at stopping rapidly progressing paralysis. While researchers have established that gene therapy stimulates regulatory T cells in the liver, Hoffman said little else is known about the detailed mechanics of how that process works.

Before the therapy can be tested in humans during a clinical trial, further research involving other preclinical models will be needed, Hoffman said. Nonetheless, Hoffman said he is extremely optimistic that his gene immunotherapy could someday be an effective treatment in humans suffering from multiple sclerosis and other autoimmune diseases. The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide.

Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. Sequences of similar lengths are preferably aligned using a global alignment algorithms e. Needleman Wunsch which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm e. Smith Waterman.

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Sequences may then be referred to as "substantially identical" or "essentially similar" when they when optimally aligned by for example the programs GAP or BESTFIT using default parameters share at least a certain minimal percentage of sequence identity as defined below. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length full length , maximizing the number of matches and minimizing the number of gaps.

A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths.

Gene Therapy

Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Following a single injection into the brain parenchyma, expression of the transgene is local and remains mainly restricted to the infused area.

Development of Gene Therapy for Neurological Disorders - Fredric P Manfredsson - Discovery Medicine

Using the CSF as a method for delivery of the viral vector, a larger area can be reached. The current study shows that using an AAV-5 vector, it is possible to reach this goal of transduction over a larger area in the CNS. The treatment was well tolerated as no adverse clinical signs and no obvious neuronal loss were observed.

Both neuronal and glial cells were transduced using a ubiquitous CAG promoter and no transgene expression was observed outside the CNS. Using cell specific promoters, such as the GFAP promoter or synapsin-1 promoter, one can direct the expression to a specific population.

We have shown that areas of the cortex, cerebellum and subventricular zone show expression of the transgene in both neurons and glial cells. In the spinal cord, motorneuron transduction was prevalent as well as transduction of neurons in the dorsal root ganglia. These results show that the method of CNS-mediated delivery can be used to deliver genes for a gene therapeutic approach. Indications that could be helped by this could be: motorneuron diseases, sensory related indications and a variety of other neurological indications.

In a first aspect, the present invention relates to an adeno-associated virus AAV gene therapy vector for use as a medicament in a mammalian subject. The invention pertains to an AAV gene therapy vector as indicated above, for use as a medicament. That is to say, the invention provides an AAV gene therapy vector of the invention for use in the method of treatment of the human or animal body by therapy.

The invention further pertains to an AAV gene therapy vector as herein defined above, for use in the treatment of a disorder, such as for example a motorneuron disease, a sensory related indication and a variety of other neurological disorders. Accordingly, the invention relates to an AAV gene therapy vector of the invention for use in the preparation of medicament for use in a method of treatment of a disorder, such as a disorder further defined herein. Viruses of the Parvoviridae family are small DNA animal viruses.

The family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. Members of the subfamily Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirus.

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.

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.

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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. 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.

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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.


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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. All Rights Reserved. NeurologyLive does not provide medical, diagnostic, or treatment advice. Videos Clinical Focus Conferences More. Currently Viewing. Disease Management on the Horizon in Alzheimer Disease. Preclinical trials and success stories suggest that much is riding on vector-based therapies for the treatment of rare neurological conditions.

By: Nicola Davies, PhD. Advances in gene therapy technology are showing increasing promise for the effective treatment of rare neurological disorders. The most recent and notable demonstration of this is the FDA approval of Zolgensma for spinal muscular atrophy SMA , with more advancements on the horizon for other disorders such as Rett syndrome RTT , a rare neurodevelopmental disorder that exhibits X chromosome linkage and is the predominant cause of severe mental retardation in girls.

Incidence is approximately 1 in 10, female births worldwide.