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Gene Therapy: Direct Viral Delivery
Gene Therapy: Direct Viral Delivery
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Gene Therapy: Direct Viral Delivery B K Kaspar, The Ohio State University, Columbus, OH, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction As populations age, the burden of health-related diseases due to nervous system disorders rises. Given the challenge to deliver drugs to the complex structure and diverse cell types of the nervous system, new therapeutic approaches are being investigated. Recent progress in molecular biology and virology has allowed tremendous advancements in delivery of recombinant DNA to the brain. This article provides an update on the status of direct viral gene delivery and examines the challenges to advance this approach as a therapeutic modality. Direct viral injections focused on treating or curing acquired and inherited disorders can be utilized through a variety of approaches, including (1) delivery of a gene in excess (such as a trophic factor), (2) suppression of a gene (via antisense or small interfering RNA), (3) swapping an abnormal gene with a normal gene through homologous recombination, (4) controlling the regulation of endogenous gene expression (via transcription factor or zinc finger approaches), or (5) modulating immune defense mechanisms by either activating or suppressing immune function. Furthermore, direct viral injection can be used to generate animal models of mutant or toxic gene expression or, using loxP technology, conditional gene deletions. There have been tremendous advances in the development of recombinant viral vectors that transduce postmitotic cells of the central nervous system with no associated toxicity. Among the two most commonly used are recombinant adeno-associated virus (AAV) vectors and recombinant lentiviral vectors. Initial studies in rodents have shown that neurons are the predominant cells that are transduced, with little transduction of other central nervous system (CNS) cell types. Both adeno-associated virus and lentiviral vectors have also been utilized in larger species, including in the monkey CNS, demonstrating a neural tropism. Over the years, it has been shown that these vectors are very nontoxic, with little to no activation of astrocytes or immune reactions against the vector. Additionally, long-term gene expression has been achieved, lasting well over 1 year post-infection with no evidence of cell death. Utilizing precise maps of the brain and spinal cord, direct injection provides an ability to specifically target regions of the brain. The dynamics of vector
spread have been studied, and, given the structure of the target and well as the type of virus; vector spread may be confined or may spread several millimeters. It has become apparent in many studies that virus may be transported in a retrograde and anterograde fashion, thereby targeting multiple areas of the CNS. Therefore, in many situations a direct injection will target multiple projection pathways.
Delivery Platforms Numerous viral-mediated gene delivery platforms exist, each having particular advantages and disadvantages. Two of the most common platforms utilized to target nondividing cells, such as neurons, are adeno-associated virus and lentiviral vectors. These two vectors are attractive since they deliver genes into postmitotic cells and have demonstrated longterm gene transduction with limited or no immune response to the vector. Retroviral vectors are used for direct injection into the brain to target dividing cells, such as astrocytes or neural progenitor cells. It has recently been shown that retroviral vectors could transduce neural progenitor cells in different regions of the brain, with long-term gene expression. In addition, adenoviral and herpes simplex viruses are advantageous for transducing all cell types in the CNS very efficiently. Both of these vectors have the potential to induce an immune response, but new modified, or ‘gutless,’ versions of the viruses have bypassed many of the immune responses. Adenoviral and herpes simplex vectors are commonly used in many brain-related cancer studies. The following sections provide a brief description of the various viruses that are used most commonly for direct injections into the brain. Adeno-Associated Viral Vectors
The AAV is a member of the genus Dependovirus of the family Parvoviridae, among the smallest of DNA animal viruses, consisting of a virion particle approximately 25 nm in diameter and composed of an icosohedron-structured protein capsid outer coat. The viral genome is linear, consisting of a singlestranded DNA molecule of approximately 4.7 kb which contains two open reading frames (ORFs) flanked by 145 bp inverted terminal repeats (ITRs). AAV requires adenovirus or herpes virus as helper virus to initiate and complete its viral life cycle of rescue, replication, and packaging. Recombinant AAV-based vectors have the viral genome deleted, other than the ITRs, allowing for insertion of vector
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construct to a maximum of approximately 4.9 kb. In recombinant form, most of the AAV vectors persist as concatemers, forming linear and circular forms, which lead to long-term persistence of the viral genome in transduced cells. Specifically, to make these vectors recombinant, the rep- and cap-coding regions are removed and a promoter with a respective cDNA and polyadenlyation signal is cloned in between the two ITRs. For viral production, this recombinant AAV plasmid, along with a trans rep and cap plasmid and helper adenovirus or adenoviral plasmid, is transfected into cells such as human embryonic kidney (HEK293) carcinoma cells. Viral production consists of lysing cells followed by gradient ultracentrifugation and/or column chromatography. Vector preparations of titers between 1012 and 1013 are now routinely produced. Viral packaging efficiency and purity are important for optimal transduction in vivo. Of importance, AAV vectors can target postmitotic cells such as neurons. Most serotypes are neurotropic; however, some have achieved astrocyte gene transfer. Most regions of the brain have been transduced by AAV, with expression lasting long term in rodents as well as nonhuman primates. Currently, the most widely utilized serotype of AAV has been based on AAV-2, which utilizes the heparan sulfate proteoglycan as the primary receptor as well as avb5 integrin and fibroblast growth factor receptor 1 (FGF1R) as coreceptors. This allows a wide host range of tissue and cellular types. Other serotypes have shown great promise for expression in ependymal cells as well as various regions of the brain. In sum, recombinant AAV vectors offer high promise for direct in vivo injection. This is due to the high stability of the virus, including an ability to inject highly concentrated stocks. AAV vectors have shown the ability to infect a wide spectrum of cells, including postmitotic cells, leading to long-term expression. Furthermore, AAV vectors are nonpathogenic, induce no etiological factors, and have been safely demonstrated in a number of clinical trials. However, AAV has a smaller packaging capacity compared to other vectors and requires several weeks to reach the maximal level of gene expression. Additionally, the scale of virus production and the purification processes remain a challenge, though significant progress is being made. New approaches using self-complementary vectors may offer some benefits for improving the timing and level of expression in the brain. Retroviral
Historically, retrovirus-mediated gene transfer methods have been the most commonly used in gene transfer techniques, based on the Moloney murine leukemia
virus (MoMuLV) as the backbone. Retroviruses target dividing cells and involve integration of the proviral DNA into the infected cell, allowing stable transmission to subsequent generations of the infected cells. Retroviruses have several advantages, including the ease of manipulation, the ability to clone relatively large genes (up to 7 kb), and the ability to infect a variety of cell types from various species. The majority of viral genes can be removed from the vectors, requiring regions in and around the long terminal repeats (LTRs) for packaging the RNA genome into the viral coat and allowing reverse transcription and integration. Since the proteins necessary for the viral replication can be provided in trans, replication-defective viruses can be generated easily. Production of retroviral vectors typically utilizes packaging and producer cell lines in which the viral gag, pol, and env proteins are expressed. Given the ability to stably infect dividing cells, current direct injections of retroviral vectors to the brain have focused on tumor cell injections as well as direct injections into regions of neurogenesis to target neural progenitor cells. This approach to label neural progenitor cells has provided a very useful tool to investigate the neurobiology and histology of neural progenitor cells in vivo. Furthermore, this approach may be very useful to target these cells for therapeutic options in a number of diseases. There are several problems with retroviruses, such as stability (very short half-life of the virus) as well as the difficulty in making concentrated high-titer stocks. Furthermore, promoter shutdown has been an issue; however, advances in vector and promoter design have proved successful. Lentiviral
Since conventional retroviruses only infect dividing cells, considerable attention has been directed to vectors based on the human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), species of the genus Lentivirus, which infect postmitotic cells. A generation of HIV-based retroviral vectors that are not limited to infection of replicating cells has gained considerable use as a means to deliver genes to the CNS. Unlike other retroviruses that rely on the breakdown of the nuclear membrane and passive diffusion into the nucleus during cell division to transport their genetic information, HIV can be actively transported to the host cell nucleus by the cellular nuclear transport mechanism. This HIV-based expression system can efficiently transfer genes into nondividing cells. The HIV-based vector has been developed through several generations, reducing the potential for recombination. Extensive studies selectively removing the HIV virulence genes (tat, vif, vpu, vpr, and nef ) have
Gene Therapy: Direct Viral Delivery
demonstrated that these new generations are helpervirus free. Lentiviruses have an advantage in that larger transgenes may be cloned into the vector, versus AAV vectors. Packaging of lentiviruses is very similar to that of retroviruses, in which the gal–pol gene is used, along with the rev gene in some packaging systems. In addition, the packaging system includes the pseudotyping of the envelopes with other viruses, such as the vesicular stomatitis virus glycoprotein (VSVG). One drawback in lentiviruses is that production in large quantities for in vivo studies remains cumbersome and also that purification techniques have not been fully developed for highly purified vectors. Recent advances in column chromatography may aid in the enhancing lentiviral purification. For human studies, it may be useful to develop vectors with nonhuman lentiviruses, such as EIAV, bovine immunodeficiency virus, or simian immunodeficiency virus.
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In sum, adenoviral vectors can be produced in high titers that hold a large amount of foreign DNA with the ability to infect postmitotic and dividing cells. The major disadvantage is that adenoviral proteins are highly immunogenic and many people have circulating antibodies and cellular immune responses toward the virus. Other drawbacks are that transgene expression is relatively unstable because the virus remains episomal, and that the virus may be toxic at high doses. The recent development of new generations of adenoviral vectors might overcome the toxicity and immunogenicity issues experienced with the previously used adenoviral vectors in the nervous system. Helper-dependent (‘gutless’) adenoviral vectors have shown reduced toxicity and larger cloning capacity compared to the standard adenoviruses. However, these gutless adenoviral systems are difficult to produce in large quantities. Herpes Simplex Virus
Adenoviral
Adenoviral vectors have been one of the most widely used viruses in gene delivery. They are nonenveloped, double-stranded (ds) DNA viral vectors with a packaging capacity of approximately 35 kb. Over 50 different adenoviral serotypes exist, grouped into six species. Recombinant adenoviral vectors are commonly derived from types 2 and 5. Adenoviruses of these serotypes have not been associated with human malignancies. The majority of these vectors are E1 gene-deleted to render the virus replication deficient, along with E3 deletions to allow greater packaging space. Most of the serotypes are able to enter most cells via the cellular coxsackie/adenovirus receptor (CAR), for attachment, and utilize av integrins for internalization. This ability is indeed a major strength of adenoviruses, enabling a broad tissue tropism and infection of many cell/tissue types, with a very fast expression profile. In addition, large genes can be inserted relatively easy and adenoviral vectors are produced with relative ease at high titers and are easily purified. Virus is produced typically in HEK293 cells that stably express the E1A and E1B genes, allowing for replication and packaging. Transgene expression from first-generation vectors is very strong, but is reduced and abolished 2–3 weeks following direct injection. These recombinant adenoviral approaches have been utilized for many direct brain injections for tumor cell killing. The adenoviruses are recognized by the immune system and therefore have problems with long-term gene expression. Second-generation vectors have been used in an attempt to decrease immune responses to the vector, by deleting early regions involved in DNA replication, such as the E2A, E2B, and E4 regions.
Recombinant herpes simplex virus (HSV-1) is a double-stranded virus that has been produced as a recombinant vector system based on a natural neurotropic virus. The two types that are being exploited for in vivo injection are the herpes virus amplicon and the recombinant virus. The virus has a large cloning capacity and has been shown to remain in a latent state within neurons. Recombinant HSV-1 vectors are created by deleting the nonessential viral genes and replacing them with a transgene or a construct of interest (up to approximately 50 kb). HSV-1 has a wide ability to infect different cells and tissues. The virus has the capacity to retrogradely transport from remote sites, affording HSV-1 the ability to target CNS sensory and motor neurons in a highly efficient manner. There is also evidence to support transsynaptic passage from neuron to neuron. Most neurons are efficiently infected by HSV-1 vectors; however, HSV-1 is not solely a neurotropic virus, and in fact it will infect astrocytes and oligodendrocytes. HSV-1 vectors have been predominantly utilized experimentally for tumor cell killing. There is, however, potential to develop an immune response to the vector, and at present, sustained transgene expression has been difficult to achieve.
Control of Vector Systems There are numerous methods to gain control of vector systems. One method is to engineer cell type-specific promoters into the recombinant vector. In this case, known cell type promoters are cloned in front of the recombinant transgene, which leads to specific cellular expression. Numerous promoters have been utilized, including promoters specific for neurons and
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for specific subtypes of neurons (such as motor neurons), as well as astrocytes. In addition to cell typespecific expression, numerous advances have allowed for regulated transgene expression. In this case, transgene expression can be turned on and off as well as regulated as to the level of expression. Technology of regulatable systems is growing, and new developments are under way that are currently being tested in a variety of vector systems. Virologists have also utilized another method to gain control of vector systems: each viral particle is coated or encapsidated, allowing researchers to modify the outer coat or capsid. Many viruses have numerous serotypes and are species specific. It has been determined that these different serotypes display different transduction profiles. Some serotypes work better than others in neurons. In addition, numerous viral vectors can be pseudotyped with various proteins that allow the virus to infect cells more efficiently. Also, specific engineering can be employed on the capsid by inserting peptides or mutating the capsid regions in specific or nonspecific regions to alter viral tropism and transduction properties. The control of vector systems in the CNS is important for a number of reasons. First, any therapeutic approach will necessitate the therapy being delivered to the proper cell type at the right dosage. Furthermore, while the brain is normally immune privileged due to the blood–brain barrier, there is considerable evidence that immune responses to viral vectors need to be considered in the design of direct viral injections. In the case of AAV, approximately 80% of the human population is seropositive for antibodies to wild-type AAV 2 capsid protein. The presence of neutralizing antibodies may prevent effective infection of this serotype. While it was originally thought that the blood– brain barrier would prevent peripheral immune responses, it has been shown that direct viral delivery locally disrupts the blood–brain barrier. Vector redosing is not possible with the same serotype. With the demonstration that viral vectors are highly efficient for delivering genes, with long-term gene expression in rodents exceeding 18 months and over 3.5 years in primate brain, it is apparent that gene delivery may be a useful modality for the treatment of progressive neurodegenerative disorders. The following sections describe recent preclinical research of direct viral delivery in a number of degenerative disorders.
Diseases of the CNS While not exclusive to all disorders, direct viral injection to the nervous system has been studied in numerous diseases and injuries, including Alzheimer’s
disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, lysosomal storage disorders, and Huntington’s disease (HD). Alzheimer’s Disease
Affecting approximately 20 million people worldwide, AD is the most prevalent neurodegenerative disease. The disease is characterized by progressive cell death in cortical structures, with progressive memory loss, impaired judgment, confusion, and personality changes. The disease is characterized by a neuropathological component of accumulated amyloid beta (Ab) plaques and neurofibrillary tangles throughout the brain. Since AD affects multiple regions of the brain, including widespread structures such as the cortex, direct viral delivery has been difficult. To date, there are no effective drugs for AD, although several inhibitors of acetylcholinesterase are prescribed. New strategies have focused on the use of trophic factors and antiapoptotic factors to target the cholinergic neurons of the basal forebrain. Nerve growth factor (NGF) is the most potent neurotrophic factor for cholinergic neurons and has been tested by direct viral delivery of NGF to spare cholinergic neurons of the basal forebrain in animal models. Furthermore, NGF has been shown to increase acetylcholine levels, which leads to improved memory, and is therefore a major candidate being pursued in the clinic. New viral approaches have focused on using natural peptidases that hydrolyze Ab. Using a number of different viral vectors, neprilysin has shown promise in decreasing Ab levels in AD mice. Another approach at reducing amyloid plaques has focused on the use of developing antibodies to clear the plaques. The use of vectors that express Ab has induced autoantibodies that have been shown to vaccinate against and thereby reduce the levels of Ab. Still another method using viral vectors has focused on the Ab proteolytic pathway to prevent the toxic form of Ab. Using a small interfering RNA (siRNA) approach against a protease (BACE-1) in the cleavage of amyloid precursor protein, cleavage has also shown significant promise as a therapy. Delivering the therapy in AD to the right cells in a widespread manner and utilizing the most efficacious genes are current challenges in AD research. Parkinson’s Disease
PD is one of the most studied neurodegenerative diseases and is the second most common disorder following AD, with symptoms manifesting at approximately 60 years of age. This neurological disorder is due to a gradual degeneration of dopamine-containing neurons in the substantia nigra, leading to the loss of dopamine transmission in the striatum. In fact, when
Gene Therapy: Direct Viral Delivery
symptoms of Parkinson’s appear, approximately 80% of striatal dopamine is lost. The loss of striatal dopamine leads to a resting tremor, bradykinesia, and akinesia. The etiology of the idiopathic form of the disease is largely unknown, although there are certain forms of the disorder with genetic components. Currently the only pharmaceutical therapy is L-dopa (L-3,4-dihydroxyphenylalanine), a precursor of the neurotransmitter dopamine. As the disease progresses over 3–5 years, this treatment becomes ineffective. Numerous studies have focused on direct gene delivery to deliver factors to prevent the neurons from dying, or to serve in dopamine replacement strategies. Several studies have utilized antiapoptotic factors or neurotrophic factors, including glial-derived neurotrophic factor for potent neuroprotection and regeneration of dopaminergic neurons. Additionally, there have been studies for striatal L-dopa for dopamine delivery via intrastriatal gene delivery. Numerous genes have been employed, including those encoding tyrosine hydroxylase (TH), GTP-cyclohydrolase 1 (GTPCH-1; for tetrahydrobiopterin availability), and aromatic amino acid decarboxylase (AADC; to produce dopamine). Still another approach has targeted the subthalamic nucleus to modulate basal ganglia during the nigrostriatal dopamine degeneration. In this therapy, direct injection into the subthalamic nucleus of glutamic acid decarboxylase (GAD) has been tested to increase the inhibitory neurotransmitter g-aminobutyric acid (GABA). All of these approaches are showing promise in rodent and nonhuman primate models of PD using direct viral injection into the CNS. Clinical trials in PD A neurotrophic strategy is currently in clinical trials to treat PD. This approach has utilized a neurotrophic factor, neurturin, to restore lost motor function and protect against further losses of dopaminergic neurons. Using an AAV encoding this gene, the virus is delivered into dopamine neuron-rich regions of the brain. Phase I safety trials in 12 patients with advanced PD were injected with the virus into the putamen at two different doses. Patients that received the low dose of virus demonstrated approximately a 36% improvement in the Unified Parkinson’s Disease Rating Score (UPDRS) while off their current drug therapies, which was detected at 9 months post-gene delivery. The group of patients receiving a fourfold higher dosage experienced similar improvements at 6 months postinjection. These patients had persistent improvements throughout the 1-year analysis. No untoward or serious adverse events were noted in the study, demonstrating that gene delivery was safe and well tolerated over long periods of time. Self-reports from patients demonstrated a 50% reduction in the time when normal
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medication was ineffective and a doubling of time without dyskinesias while on medication. Currently, a phase II trial of this treatment is underway, enrolling 51 patients with advanced PD, and will be studied for 12 months for safety and efficacy. A second trial using AAV virus is currently underway to evaluate the safety and efficacy of a therapy designed to restore and extend the therapeutic effectiveness of L-dopa, the precursor to dopamine. This vector encodes aromatic amino acid decarboxylase and is currently administered bilaterally to the putamen of patients. As of 2006, four patients had received treatment. Positron emission tomography (PET) anlaysis showed an increase in the ratio of L-dopa to dopamine, demonstrating an increase in the AADC tracer fluoro-L-m-tyrosine (FMT) uptake at 1 month in all four patients following gene delivery, with slightly less uptake at 6 months. To date, there has been no report on changes in the average UPDRS motor score. Further recruitment is currently under way to evaluate this clinical study. Another trial that has demonstrated safety and function of AAV has involved 11 men and one woman with PD, a disease noted for substantial reduction in the activity and amount of GABA in the brain; GABA is a major inhibitory brain neurotransmitter that suppresses excessive neuronal firing. In this trial approach, using an adeno-associated viral vector, the glutamic acid decarboxylase (GAD) gene was injected into a single site of the brain, the subthalamic nucleus, that is a key regulatory center within the motor circuit. In this procedure, only one side of the brain was injected with virus. Using the UPDRS and positron emission tomography scans, the investigators monitored patients for safety and the potential efficacy. They found that the injection was safe and well tolerated, and no adverse events were reported during the trial over 1 year. Interestingly, there was a 25–30% improvement in the UPDRS scores in patients taken off the standard drug therapies for a period of time, to evaluate effects of the gene therapy treatment alone. Similar improvements were found even while patients were on their normal medications. These improvements persisted over the course of the trial. Trends noted on reduced medication linked dyskinesias and improvements in daily living, though the data did not reach statistical significance. Furthermore, PET scans showed that the treated side of the brain had more normal levels of activity than did the nontreated side. Larger, efficacy-driven trials are planned to evaluate this therapy. Amyotrophic Lateral Sclerosis
Also known as Lou Gehrig’s disease, ALS is a degenerative disease of the motor system affecting upper
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and lower motor neurons of the motor cortex and spinal cord. The disease is characterized by loss of skeletal muscle control, ultimately resulting in death due to loss of muscles involved in respiration. Currently there are no effective treatments for this disease, but significant progress and promise have been demonstrated by gene therapy approaches. Numerous strategies have evaluated viral-mediated gene delivery of neurotrophic factors – glial-derived neurotrophic factor (GDNF), insulin-like growth factor1 (IGF-1), and vascular endothelial growth factor (VEGF) – as well as antiapoptotic genes using numerous viral vectors, including adenoviruses, AAV, and lentiviruses. In addition, there are developments to deliver antisense or siRNAs directed against one of the known genetic mutations accounting for the familial form of the disease in which patients have mutations within the superoxide dismutase 1 (SOD1) gene. Several different approaches have been utilized to deliver a therapeutic gene to the spinal cord. These approaches have utilized the concept of remote delivery by injecting the therapeutic vector intramuscularly; the virus is taken up by the axon terminal and transported in a retrograde fashion to the motor neuron cell body. This retrograde transport approach of the viral vector was accomplished using adenoviral and adeno-associated viral vectors as well as rabies glycoprotein pseudotyping of lentiviral vectors. Other approaches have investigated direct viral delivery by intraparenchymal delivery to the spinal cord. These studies have all showed the promise to significantly delay disease onset and/or progression in mouse models of the disease. Several of the approaches are being developed for clinical translation. Ultimately, as further genetic analysis of patients leads to new targets and mechanisms of the disease, the efforts gained in targeting spinal cord motor neurons and the surrounding cellular environment will lead to more efficacious therapies. Lysosomal Storage Disorders
Over 40 known lysosomal disorders result from deficient enzymes, leading to accumulation of cellular proteins in the lysosomes. A deficiency of lysosomal acid hydrolases results in several lysosomal disorders, including mucopolysaccharidosis type VII (MPS VII), Niemann–Pick A, MPS IIIB, Krabbe disease, and Batten disease. Current therapies include enzyme replacement strategies. Human therapy for prenatally determined lysosomal disorders requires administration of the missing enzyme postnatally. Approaches to gene therapy have tested numerous enzyme replacement strategies utilizing viral vectors, and new approaches are investigating minimally invasive, yet
widespread gene delivery to the CNS in addition to delivering the gene to peripheral organs. The CNS is targeted to treat the cognitive neural problems associated with the disease. A clinical trial for Batten disease is currently underway to transfer a correct version of the ceroid lipofuscinosis neuronal 2 (CLN2) gene into infants. Huntington’s Disease
HD is a fatal neurodegenerative disease classified by trinucleotide repeats of CAG in the first exon of the HD gene. These polyglutamine expansions require a specific length to trigger neurodegeneration, in which the mutant protein forms aggregate, leading to loss of GABA spiny neurons in the caudate putamen. The disease leads to cognitive defects and motor abnormalities. Trophic factor support utilizing a number of viral vectors is currently being tested to protect against the GABAergic cell loss. Furthermore, new approaches utilizing short-hairpin RNAs (shRNAs) to target the mutant huntington gene has shown significant promise for a therapeutic modality in this disease. Given that a number of diseases of the CNS are due to the expansion of polyglutamine repeats, this directed approach for silencing the mutant protein may be further applicable to other diseases. Proper delivery to target the affected cells in the disease and the optimal timing of delivery are challenges that researchers in the field are currently developing.
Conclusion In addition to use in therapeutic approaches to the diseases discussed in this article, gene therapy by direct viral injection has shown significant promise for the treatment of brain disorders such as Canavan’s disease, and in treating pain, epilepsy, stroke, and spinal cord injury; besides, there is interest in direct viral injection in a wide spectrum of studies on treating cancers of the brain, including neuroblastoma and glioblastoma. Utilization of gene therapy by direct viral injection for nervous system disorders has the potential to revolutionize medicine in the near future. Indeed, there have been striking preclinical results demonstrating that viral vectors may be the only feasible therapeutic modality to target a specific region of the brain, or to deliver a therapy that can get past the blood–brain barrier. Many of these preclinical studies warrant clinical development. As additional genetic understanding for brain diseases develops, the ability to utilize the optimal vectors with the right gene, by the optimal injection parameter, will ultimately result in therapeutic benefit.
Gene Therapy: Direct Viral Delivery
Truly one can see the challenges and struggles in the field of gene delivery as similar to those faced in the dramatic development of open heart surgery, when the early studies resulted in scientific challenges that were far beyond those ever imagined, including the loss of the earliest treated patients. The field of gene therapy, in particular as it relates to neuroscience, has recruited a diversity of specialists, ranging from molecular biologists to virologists, anatomists, physicians, and biotechnologists, to work together to revolutionize healthcare and ultimately to unburden patients from the effects of disease and injury of the nervous system. As clinical development of gene therapy ensues, the field will take the lessons learned and apply them to evolve genetic medicines to use in standard practice. See also: Cell Replacement Therapy: Parkinson’s Disease; Cell Replacement Therapy: Mechanisms of Functional Recovery; Cell Replacement Therapy for Huntington’s Disease; Enteric Nervous System: Neurotrophic Factors; Gene Therapy: Genetically Modified Cells; GFL Neurotrophic Factors: Physiology and Pharmacology; Neurotrophic Factor Therapy: GDNF and CNTF; Neurotrophic Factor Therapy: NGF, BDNF and NT3; Viral Vectors in the CNS.
Further Reading Bankiewicz KS, Leff SE, Nagy D, et al. (1997) Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson’s disease. Experimental Neurology 144: 147–156. Bohn MC (1999) A commentary on glial cell line-derived neurotrophic factor (GDNF). From a glial secreted molecule to gene therapy. Biochemical Pharmacology 57: 135–142. Bruijn LI and Cudkowicz M (2006) Therapeutic targets for amyotrophic lateral sclerosis: Current treatments and prospects for more effective therapies. Expert Review of Neurotherapeutics 6: 417–428. Burger C, Gorbatyuk OS, Velardo MJ, et al. (2004) Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell
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tropism after delivery to different regions of the central nervous system. Molecular Therapy 10: 302–317. Carlsson T, Bjorklund T, and Kirik D (2007) Restoration of the striatal dopamine synthesis for Parkinson’s disease: Viral vector-mediated enzyme replacement strategy. Current Gene Therapy 7: 109–120. Denovan-Wright EM and Davidson BL (2006) RNAi: A potential therapy for the dominantly inherited nucleotide repeat diseases. Gene Therapy 13: 525–531. Federici T and Boulis NM (2006) Gene-based treatment of motor neuron diseases. Muscle & Nerve 33: 302–323. Jakobsson J and Lundberg C (2006) Lentiviral vectors for use in the central nervous system. Molecular Therapy 13: 484–493. Kaplitt MG, Feigin A, Tang C, et al. (2007) Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: An open label, phase I trial. Lancet 369: 2097–2105. Kaspar BK, Llado J, Sherkat N, et al. (2003) Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science 301: 839–842. Naldini L, Blomer U, Gallay P, et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263–267. Ryan DA and Federoff HJ (2007) Translational considerations for CNS gene therapy. Expert Opinion on Biological Therapy 7: 305–318. Samulski RJ (2003) AAV vectors, the future workhorse of human gene therapy. Ernst Schering Research Foundation Workshop 43: 25–40. Sands MS and Davidson BL (2006) Gene therapy for lysosomal storage diseases. Molecular Therapy 13: 839–849. St George JA (2003) Gene therapy progress and prospects: Adenoviral vectors. Gene Therapy 10: 1135–1141. Suhr ST and Gage FH (1993) Gene therapy for neurologic disease. Archives of Neurology 50: 1252–1268. Suhr ST and Gage FH (1999) Gene therapy in the central nervous system: The use of recombinant retroviruses. Archives of Neurology 56: 287–292. Tashiro A, Zhao C, and Gage FH (2006) Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo. Nature Protocols 1: 3049–3055. Tyler CM, Wuertzer CA, Bowers WJ, et al. (2006) HSV amplicons: Neuro applications. Current Gene Therapy 6: 337–350. Wu Z, Asokan A, and Samulski RJ (2006) Adeno-associated virus serotypes: Vector toolkit for human gene therapy. Molecular Therapy 14: 316–327.
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Gene Therapy: Direct Viral Delivery B K Kaspar, The Ohio State University, Columbus, OH, USA ã 2009 Elsevier Ltd. All rights reserved.
Introduction As populations age, the burden of health-related diseases due to nervous system disorders rises. Given the challenge to deliver drugs to the complex structure and diverse cell types of the nervous system, new therapeutic approaches are being investigated. Recent progress in molecular biology and virology has allowed tremendous advancements in delivery of recombinant DNA to the brain. This article provides an update on the status of direct viral gene delivery and examines the challenges to advance this approach as a therapeutic modality. Direct viral injections focused on treating or curing acquired and inherited disorders can be utilized through a variety of approaches, including (1) delivery of a gene in excess (such as a trophic factor), (2) suppression of a gene (via antisense or small interfering RNA), (3) swapping an abnormal gene with a normal gene through homologous recombination, (4) controlling the regulation of endogenous gene expression (via transcription factor or zinc finger approaches), or (5) modulating immune defense mechanisms by either activating or suppressing immune function. Furthermore, direct viral injection can be used to generate animal models of mutant or toxic gene expression or, using loxP technology, conditional gene deletions. There have been tremendous advances in the development of recombinant viral vectors that transduce postmitotic cells of the central nervous system with no associated toxicity. Among the two most commonly used are recombinant adeno-associated virus (AAV) vectors and recombinant lentiviral vectors. Initial studies in rodents have shown that neurons are the predominant cells that are transduced, with little transduction of other central nervous system (CNS) cell types. Both adeno-associated virus and lentiviral vectors have also been utilized in larger species, including in the monkey CNS, demonstrating a neural tropism. Over the years, it has been shown that these vectors are very nontoxic, with little to no activation of astrocytes or immune reactions against the vector. Additionally, long-term gene expression has been achieved, lasting well over 1 year post-infection with no evidence of cell death. Utilizing precise maps of the brain and spinal cord, direct injection provides an ability to specifically target regions of the brain. The dynamics of vector
spread have been studied, and, given the structure of the target and well as the type of virus; vector spread may be confined or may spread several millimeters. It has become apparent in many studies that virus may be transported in a retrograde and anterograde fashion, thereby targeting multiple areas of the CNS. Therefore, in many situations a direct injection will target multiple projection pathways.
Delivery Platforms Numerous viral-mediated gene delivery platforms exist, each having particular advantages and disadvantages. Two of the most common platforms utilized to target nondividing cells, such as neurons, are adeno-associated virus and lentiviral vectors. These two vectors are attractive since they deliver genes into postmitotic cells and have demonstrated longterm gene transduction with limited or no immune response to the vector. Retroviral vectors are used for direct injection into the brain to target dividing cells, such as astrocytes or neural progenitor cells. It has recently been shown that retroviral vectors could transduce neural progenitor cells in different regions of the brain, with long-term gene expression. In addition, adenoviral and herpes simplex viruses are advantageous for transducing all cell types in the CNS very efficiently. Both of these vectors have the potential to induce an immune response, but new modified, or ‘gutless,’ versions of the viruses have bypassed many of the immune responses. Adenoviral and herpes simplex vectors are commonly used in many brain-related cancer studies. The following sections provide a brief description of the various viruses that are used most commonly for direct injections into the brain. Adeno-Associated Viral Vectors
The AAV is a member of the genus Dependovirus of the family Parvoviridae, among the smallest of DNA animal viruses, consisting of a virion particle approximately 25 nm in diameter and composed of an icosohedron-structured protein capsid outer coat. The viral genome is linear, consisting of a singlestranded DNA molecule of approximately 4.7 kb which contains two open reading frames (ORFs) flanked by 145 bp inverted terminal repeats (ITRs). AAV requires adenovirus or herpes virus as helper virus to initiate and complete its viral life cycle of rescue, replication, and packaging. Recombinant AAV-based vectors have the viral genome deleted, other than the ITRs, allowing for insertion of vector
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construct to a maximum of approximately 4.9 kb. In recombinant form, most of the AAV vectors persist as concatemers, forming linear and circular forms, which lead to long-term persistence of the viral genome in transduced cells. Specifically, to make these vectors recombinant, the rep- and cap-coding regions are removed and a promoter with a respective cDNA and polyadenlyation signal is cloned in between the two ITRs. For viral production, this recombinant AAV plasmid, along with a trans rep and cap plasmid and helper adenovirus or adenoviral plasmid, is transfected into cells such as human embryonic kidney (HEK293) carcinoma cells. Viral production consists of lysing cells followed by gradient ultracentrifugation and/or column chromatography. Vector preparations of titers between 1012 and 1013 are now routinely produced. Viral packaging efficiency and purity are important for optimal transduction in vivo. Of importance, AAV vectors can target postmitotic cells such as neurons. Most serotypes are neurotropic; however, some have achieved astrocyte gene transfer. Most regions of the brain have been transduced by AAV, with expression lasting long term in rodents as well as nonhuman primates. Currently, the most widely utilized serotype of AAV has been based on AAV-2, which utilizes the heparan sulfate proteoglycan as the primary receptor as well as avb5 integrin and fibroblast growth factor receptor 1 (FGF1R) as coreceptors. This allows a wide host range of tissue and cellular types. Other serotypes have shown great promise for expression in ependymal cells as well as various regions of the brain. In sum, recombinant AAV vectors offer high promise for direct in vivo injection. This is due to the high stability of the virus, including an ability to inject highly concentrated stocks. AAV vectors have shown the ability to infect a wide spectrum of cells, including postmitotic cells, leading to long-term expression. Furthermore, AAV vectors are nonpathogenic, induce no etiological factors, and have been safely demonstrated in a number of clinical trials. However, AAV has a smaller packaging capacity compared to other vectors and requires several weeks to reach the maximal level of gene expression. Additionally, the scale of virus production and the purification processes remain a challenge, though significant progress is being made. New approaches using self-complementary vectors may offer some benefits for improving the timing and level of expression in the brain. Retroviral
Historically, retrovirus-mediated gene transfer methods have been the most commonly used in gene transfer techniques, based on the Moloney murine leukemia
virus (MoMuLV) as the backbone. Retroviruses target dividing cells and involve integration of the proviral DNA into the infected cell, allowing stable transmission to subsequent generations of the infected cells. Retroviruses have several advantages, including the ease of manipulation, the ability to clone relatively large genes (up to 7 kb), and the ability to infect a variety of cell types from various species. The majority of viral genes can be removed from the vectors, requiring regions in and around the long terminal repeats (LTRs) for packaging the RNA genome into the viral coat and allowing reverse transcription and integration. Since the proteins necessary for the viral replication can be provided in trans, replication-defective viruses can be generated easily. Production of retroviral vectors typically utilizes packaging and producer cell lines in which the viral gag, pol, and env proteins are expressed. Given the ability to stably infect dividing cells, current direct injections of retroviral vectors to the brain have focused on tumor cell injections as well as direct injections into regions of neurogenesis to target neural progenitor cells. This approach to label neural progenitor cells has provided a very useful tool to investigate the neurobiology and histology of neural progenitor cells in vivo. Furthermore, this approach may be very useful to target these cells for therapeutic options in a number of diseases. There are several problems with retroviruses, such as stability (very short half-life of the virus) as well as the difficulty in making concentrated high-titer stocks. Furthermore, promoter shutdown has been an issue; however, advances in vector and promoter design have proved successful. Lentiviral
Since conventional retroviruses only infect dividing cells, considerable attention has been directed to vectors based on the human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), species of the genus Lentivirus, which infect postmitotic cells. A generation of HIV-based retroviral vectors that are not limited to infection of replicating cells has gained considerable use as a means to deliver genes to the CNS. Unlike other retroviruses that rely on the breakdown of the nuclear membrane and passive diffusion into the nucleus during cell division to transport their genetic information, HIV can be actively transported to the host cell nucleus by the cellular nuclear transport mechanism. This HIV-based expression system can efficiently transfer genes into nondividing cells. The HIV-based vector has been developed through several generations, reducing the potential for recombination. Extensive studies selectively removing the HIV virulence genes (tat, vif, vpu, vpr, and nef ) have
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demonstrated that these new generations are helpervirus free. Lentiviruses have an advantage in that larger transgenes may be cloned into the vector, versus AAV vectors. Packaging of lentiviruses is very similar to that of retroviruses, in which the gal–pol gene is used, along with the rev gene in some packaging systems. In addition, the packaging system includes the pseudotyping of the envelopes with other viruses, such as the vesicular stomatitis virus glycoprotein (VSVG). One drawback in lentiviruses is that production in large quantities for in vivo studies remains cumbersome and also that purification techniques have not been fully developed for highly purified vectors. Recent advances in column chromatography may aid in the enhancing lentiviral purification. For human studies, it may be useful to develop vectors with nonhuman lentiviruses, such as EIAV, bovine immunodeficiency virus, or simian immunodeficiency virus.
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In sum, adenoviral vectors can be produced in high titers that hold a large amount of foreign DNA with the ability to infect postmitotic and dividing cells. The major disadvantage is that adenoviral proteins are highly immunogenic and many people have circulating antibodies and cellular immune responses toward the virus. Other drawbacks are that transgene expression is relatively unstable because the virus remains episomal, and that the virus may be toxic at high doses. The recent development of new generations of adenoviral vectors might overcome the toxicity and immunogenicity issues experienced with the previously used adenoviral vectors in the nervous system. Helper-dependent (‘gutless’) adenoviral vectors have shown reduced toxicity and larger cloning capacity compared to the standard adenoviruses. However, these gutless adenoviral systems are difficult to produce in large quantities. Herpes Simplex Virus
Adenoviral
Adenoviral vectors have been one of the most widely used viruses in gene delivery. They are nonenveloped, double-stranded (ds) DNA viral vectors with a packaging capacity of approximately 35 kb. Over 50 different adenoviral serotypes exist, grouped into six species. Recombinant adenoviral vectors are commonly derived from types 2 and 5. Adenoviruses of these serotypes have not been associated with human malignancies. The majority of these vectors are E1 gene-deleted to render the virus replication deficient, along with E3 deletions to allow greater packaging space. Most of the serotypes are able to enter most cells via the cellular coxsackie/adenovirus receptor (CAR), for attachment, and utilize av integrins for internalization. This ability is indeed a major strength of adenoviruses, enabling a broad tissue tropism and infection of many cell/tissue types, with a very fast expression profile. In addition, large genes can be inserted relatively easy and adenoviral vectors are produced with relative ease at high titers and are easily purified. Virus is produced typically in HEK293 cells that stably express the E1A and E1B genes, allowing for replication and packaging. Transgene expression from first-generation vectors is very strong, but is reduced and abolished 2–3 weeks following direct injection. These recombinant adenoviral approaches have been utilized for many direct brain injections for tumor cell killing. The adenoviruses are recognized by the immune system and therefore have problems with long-term gene expression. Second-generation vectors have been used in an attempt to decrease immune responses to the vector, by deleting early regions involved in DNA replication, such as the E2A, E2B, and E4 regions.
Recombinant herpes simplex virus (HSV-1) is a double-stranded virus that has been produced as a recombinant vector system based on a natural neurotropic virus. The two types that are being exploited for in vivo injection are the herpes virus amplicon and the recombinant virus. The virus has a large cloning capacity and has been shown to remain in a latent state within neurons. Recombinant HSV-1 vectors are created by deleting the nonessential viral genes and replacing them with a transgene or a construct of interest (up to approximately 50 kb). HSV-1 has a wide ability to infect different cells and tissues. The virus has the capacity to retrogradely transport from remote sites, affording HSV-1 the ability to target CNS sensory and motor neurons in a highly efficient manner. There is also evidence to support transsynaptic passage from neuron to neuron. Most neurons are efficiently infected by HSV-1 vectors; however, HSV-1 is not solely a neurotropic virus, and in fact it will infect astrocytes and oligodendrocytes. HSV-1 vectors have been predominantly utilized experimentally for tumor cell killing. There is, however, potential to develop an immune response to the vector, and at present, sustained transgene expression has been difficult to achieve.
Control of Vector Systems There are numerous methods to gain control of vector systems. One method is to engineer cell type-specific promoters into the recombinant vector. In this case, known cell type promoters are cloned in front of the recombinant transgene, which leads to specific cellular expression. Numerous promoters have been utilized, including promoters specific for neurons and
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for specific subtypes of neurons (such as motor neurons), as well as astrocytes. In addition to cell typespecific expression, numerous advances have allowed for regulated transgene expression. In this case, transgene expression can be turned on and off as well as regulated as to the level of expression. Technology of regulatable systems is growing, and new developments are under way that are currently being tested in a variety of vector systems. Virologists have also utilized another method to gain control of vector systems: each viral particle is coated or encapsidated, allowing researchers to modify the outer coat or capsid. Many viruses have numerous serotypes and are species specific. It has been determined that these different serotypes display different transduction profiles. Some serotypes work better than others in neurons. In addition, numerous viral vectors can be pseudotyped with various proteins that allow the virus to infect cells more efficiently. Also, specific engineering can be employed on the capsid by inserting peptides or mutating the capsid regions in specific or nonspecific regions to alter viral tropism and transduction properties. The control of vector systems in the CNS is important for a number of reasons. First, any therapeutic approach will necessitate the therapy being delivered to the proper cell type at the right dosage. Furthermore, while the brain is normally immune privileged due to the blood–brain barrier, there is considerable evidence that immune responses to viral vectors need to be considered in the design of direct viral injections. In the case of AAV, approximately 80% of the human population is seropositive for antibodies to wild-type AAV 2 capsid protein. The presence of neutralizing antibodies may prevent effective infection of this serotype. While it was originally thought that the blood– brain barrier would prevent peripheral immune responses, it has been shown that direct viral delivery locally disrupts the blood–brain barrier. Vector redosing is not possible with the same serotype. With the demonstration that viral vectors are highly efficient for delivering genes, with long-term gene expression in rodents exceeding 18 months and over 3.5 years in primate brain, it is apparent that gene delivery may be a useful modality for the treatment of progressive neurodegenerative disorders. The following sections describe recent preclinical research of direct viral delivery in a number of degenerative disorders.
Diseases of the CNS While not exclusive to all disorders, direct viral injection to the nervous system has been studied in numerous diseases and injuries, including Alzheimer’s
disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, lysosomal storage disorders, and Huntington’s disease (HD). Alzheimer’s Disease
Affecting approximately 20 million people worldwide, AD is the most prevalent neurodegenerative disease. The disease is characterized by progressive cell death in cortical structures, with progressive memory loss, impaired judgment, confusion, and personality changes. The disease is characterized by a neuropathological component of accumulated amyloid beta (Ab) plaques and neurofibrillary tangles throughout the brain. Since AD affects multiple regions of the brain, including widespread structures such as the cortex, direct viral delivery has been difficult. To date, there are no effective drugs for AD, although several inhibitors of acetylcholinesterase are prescribed. New strategies have focused on the use of trophic factors and antiapoptotic factors to target the cholinergic neurons of the basal forebrain. Nerve growth factor (NGF) is the most potent neurotrophic factor for cholinergic neurons and has been tested by direct viral delivery of NGF to spare cholinergic neurons of the basal forebrain in animal models. Furthermore, NGF has been shown to increase acetylcholine levels, which leads to improved memory, and is therefore a major candidate being pursued in the clinic. New viral approaches have focused on using natural peptidases that hydrolyze Ab. Using a number of different viral vectors, neprilysin has shown promise in decreasing Ab levels in AD mice. Another approach at reducing amyloid plaques has focused on the use of developing antibodies to clear the plaques. The use of vectors that express Ab has induced autoantibodies that have been shown to vaccinate against and thereby reduce the levels of Ab. Still another method using viral vectors has focused on the Ab proteolytic pathway to prevent the toxic form of Ab. Using a small interfering RNA (siRNA) approach against a protease (BACE-1) in the cleavage of amyloid precursor protein, cleavage has also shown significant promise as a therapy. Delivering the therapy in AD to the right cells in a widespread manner and utilizing the most efficacious genes are current challenges in AD research. Parkinson’s Disease
PD is one of the most studied neurodegenerative diseases and is the second most common disorder following AD, with symptoms manifesting at approximately 60 years of age. This neurological disorder is due to a gradual degeneration of dopamine-containing neurons in the substantia nigra, leading to the loss of dopamine transmission in the striatum. In fact, when
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symptoms of Parkinson’s appear, approximately 80% of striatal dopamine is lost. The loss of striatal dopamine leads to a resting tremor, bradykinesia, and akinesia. The etiology of the idiopathic form of the disease is largely unknown, although there are certain forms of the disorder with genetic components. Currently the only pharmaceutical therapy is L-dopa (L-3,4-dihydroxyphenylalanine), a precursor of the neurotransmitter dopamine. As the disease progresses over 3–5 years, this treatment becomes ineffective. Numerous studies have focused on direct gene delivery to deliver factors to prevent the neurons from dying, or to serve in dopamine replacement strategies. Several studies have utilized antiapoptotic factors or neurotrophic factors, including glial-derived neurotrophic factor for potent neuroprotection and regeneration of dopaminergic neurons. Additionally, there have been studies for striatal L-dopa for dopamine delivery via intrastriatal gene delivery. Numerous genes have been employed, including those encoding tyrosine hydroxylase (TH), GTP-cyclohydrolase 1 (GTPCH-1; for tetrahydrobiopterin availability), and aromatic amino acid decarboxylase (AADC; to produce dopamine). Still another approach has targeted the subthalamic nucleus to modulate basal ganglia during the nigrostriatal dopamine degeneration. In this therapy, direct injection into the subthalamic nucleus of glutamic acid decarboxylase (GAD) has been tested to increase the inhibitory neurotransmitter g-aminobutyric acid (GABA). All of these approaches are showing promise in rodent and nonhuman primate models of PD using direct viral injection into the CNS. Clinical trials in PD A neurotrophic strategy is currently in clinical trials to treat PD. This approach has utilized a neurotrophic factor, neurturin, to restore lost motor function and protect against further losses of dopaminergic neurons. Using an AAV encoding this gene, the virus is delivered into dopamine neuron-rich regions of the brain. Phase I safety trials in 12 patients with advanced PD were injected with the virus into the putamen at two different doses. Patients that received the low dose of virus demonstrated approximately a 36% improvement in the Unified Parkinson’s Disease Rating Score (UPDRS) while off their current drug therapies, which was detected at 9 months post-gene delivery. The group of patients receiving a fourfold higher dosage experienced similar improvements at 6 months postinjection. These patients had persistent improvements throughout the 1-year analysis. No untoward or serious adverse events were noted in the study, demonstrating that gene delivery was safe and well tolerated over long periods of time. Self-reports from patients demonstrated a 50% reduction in the time when normal
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medication was ineffective and a doubling of time without dyskinesias while on medication. Currently, a phase II trial of this treatment is underway, enrolling 51 patients with advanced PD, and will be studied for 12 months for safety and efficacy. A second trial using AAV virus is currently underway to evaluate the safety and efficacy of a therapy designed to restore and extend the therapeutic effectiveness of L-dopa, the precursor to dopamine. This vector encodes aromatic amino acid decarboxylase and is currently administered bilaterally to the putamen of patients. As of 2006, four patients had received treatment. Positron emission tomography (PET) anlaysis showed an increase in the ratio of L-dopa to dopamine, demonstrating an increase in the AADC tracer fluoro-L-m-tyrosine (FMT) uptake at 1 month in all four patients following gene delivery, with slightly less uptake at 6 months. To date, there has been no report on changes in the average UPDRS motor score. Further recruitment is currently under way to evaluate this clinical study. Another trial that has demonstrated safety and function of AAV has involved 11 men and one woman with PD, a disease noted for substantial reduction in the activity and amount of GABA in the brain; GABA is a major inhibitory brain neurotransmitter that suppresses excessive neuronal firing. In this trial approach, using an adeno-associated viral vector, the glutamic acid decarboxylase (GAD) gene was injected into a single site of the brain, the subthalamic nucleus, that is a key regulatory center within the motor circuit. In this procedure, only one side of the brain was injected with virus. Using the UPDRS and positron emission tomography scans, the investigators monitored patients for safety and the potential efficacy. They found that the injection was safe and well tolerated, and no adverse events were reported during the trial over 1 year. Interestingly, there was a 25–30% improvement in the UPDRS scores in patients taken off the standard drug therapies for a period of time, to evaluate effects of the gene therapy treatment alone. Similar improvements were found even while patients were on their normal medications. These improvements persisted over the course of the trial. Trends noted on reduced medication linked dyskinesias and improvements in daily living, though the data did not reach statistical significance. Furthermore, PET scans showed that the treated side of the brain had more normal levels of activity than did the nontreated side. Larger, efficacy-driven trials are planned to evaluate this therapy. Amyotrophic Lateral Sclerosis
Also known as Lou Gehrig’s disease, ALS is a degenerative disease of the motor system affecting upper
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and lower motor neurons of the motor cortex and spinal cord. The disease is characterized by loss of skeletal muscle control, ultimately resulting in death due to loss of muscles involved in respiration. Currently there are no effective treatments for this disease, but significant progress and promise have been demonstrated by gene therapy approaches. Numerous strategies have evaluated viral-mediated gene delivery of neurotrophic factors – glial-derived neurotrophic factor (GDNF), insulin-like growth factor1 (IGF-1), and vascular endothelial growth factor (VEGF) – as well as antiapoptotic genes using numerous viral vectors, including adenoviruses, AAV, and lentiviruses. In addition, there are developments to deliver antisense or siRNAs directed against one of the known genetic mutations accounting for the familial form of the disease in which patients have mutations within the superoxide dismutase 1 (SOD1) gene. Several different approaches have been utilized to deliver a therapeutic gene to the spinal cord. These approaches have utilized the concept of remote delivery by injecting the therapeutic vector intramuscularly; the virus is taken up by the axon terminal and transported in a retrograde fashion to the motor neuron cell body. This retrograde transport approach of the viral vector was accomplished using adenoviral and adeno-associated viral vectors as well as rabies glycoprotein pseudotyping of lentiviral vectors. Other approaches have investigated direct viral delivery by intraparenchymal delivery to the spinal cord. These studies have all showed the promise to significantly delay disease onset and/or progression in mouse models of the disease. Several of the approaches are being developed for clinical translation. Ultimately, as further genetic analysis of patients leads to new targets and mechanisms of the disease, the efforts gained in targeting spinal cord motor neurons and the surrounding cellular environment will lead to more efficacious therapies. Lysosomal Storage Disorders
Over 40 known lysosomal disorders result from deficient enzymes, leading to accumulation of cellular proteins in the lysosomes. A deficiency of lysosomal acid hydrolases results in several lysosomal disorders, including mucopolysaccharidosis type VII (MPS VII), Niemann–Pick A, MPS IIIB, Krabbe disease, and Batten disease. Current therapies include enzyme replacement strategies. Human therapy for prenatally determined lysosomal disorders requires administration of the missing enzyme postnatally. Approaches to gene therapy have tested numerous enzyme replacement strategies utilizing viral vectors, and new approaches are investigating minimally invasive, yet
widespread gene delivery to the CNS in addition to delivering the gene to peripheral organs. The CNS is targeted to treat the cognitive neural problems associated with the disease. A clinical trial for Batten disease is currently underway to transfer a correct version of the ceroid lipofuscinosis neuronal 2 (CLN2) gene into infants. Huntington’s Disease
HD is a fatal neurodegenerative disease classified by trinucleotide repeats of CAG in the first exon of the HD gene. These polyglutamine expansions require a specific length to trigger neurodegeneration, in which the mutant protein forms aggregate, leading to loss of GABA spiny neurons in the caudate putamen. The disease leads to cognitive defects and motor abnormalities. Trophic factor support utilizing a number of viral vectors is currently being tested to protect against the GABAergic cell loss. Furthermore, new approaches utilizing short-hairpin RNAs (shRNAs) to target the mutant huntington gene has shown significant promise for a therapeutic modality in this disease. Given that a number of diseases of the CNS are due to the expansion of polyglutamine repeats, this directed approach for silencing the mutant protein may be further applicable to other diseases. Proper delivery to target the affected cells in the disease and the optimal timing of delivery are challenges that researchers in the field are currently developing.
Conclusion In addition to use in therapeutic approaches to the diseases discussed in this article, gene therapy by direct viral injection has shown significant promise for the treatment of brain disorders such as Canavan’s disease, and in treating pain, epilepsy, stroke, and spinal cord injury; besides, there is interest in direct viral injection in a wide spectrum of studies on treating cancers of the brain, including neuroblastoma and glioblastoma. Utilization of gene therapy by direct viral injection for nervous system disorders has the potential to revolutionize medicine in the near future. Indeed, there have been striking preclinical results demonstrating that viral vectors may be the only feasible therapeutic modality to target a specific region of the brain, or to deliver a therapy that can get past the blood–brain barrier. Many of these preclinical studies warrant clinical development. As additional genetic understanding for brain diseases develops, the ability to utilize the optimal vectors with the right gene, by the optimal injection parameter, will ultimately result in therapeutic benefit.
Gene Therapy: Direct Viral Delivery
Truly one can see the challenges and struggles in the field of gene delivery as similar to those faced in the dramatic development of open heart surgery, when the early studies resulted in scientific challenges that were far beyond those ever imagined, including the loss of the earliest treated patients. The field of gene therapy, in particular as it relates to neuroscience, has recruited a diversity of specialists, ranging from molecular biologists to virologists, anatomists, physicians, and biotechnologists, to work together to revolutionize healthcare and ultimately to unburden patients from the effects of disease and injury of the nervous system. As clinical development of gene therapy ensues, the field will take the lessons learned and apply them to evolve genetic medicines to use in standard practice. See also: Cell Replacement Therapy: Parkinson’s Disease; Cell Replacement Therapy: Mechanisms of Functional Recovery; Cell Replacement Therapy for Huntington’s Disease; Enteric Nervous System: Neurotrophic Factors; Gene Therapy: Genetically Modified Cells; GFL Neurotrophic Factors: Physiology and Pharmacology; Neurotrophic Factor Therapy: GDNF and CNTF; Neurotrophic Factor Therapy: NGF, BDNF and NT3; Viral Vectors in the CNS.
Further Reading Bankiewicz KS, Leff SE, Nagy D, et al. (1997) Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson’s disease. Experimental Neurology 144: 147–156. Bohn MC (1999) A commentary on glial cell line-derived neurotrophic factor (GDNF). From a glial secreted molecule to gene therapy. Biochemical Pharmacology 57: 135–142. Bruijn LI and Cudkowicz M (2006) Therapeutic targets for amyotrophic lateral sclerosis: Current treatments and prospects for more effective therapies. Expert Review of Neurotherapeutics 6: 417–428. Burger C, Gorbatyuk OS, Velardo MJ, et al. (2004) Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell
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tropism after delivery to different regions of the central nervous system. Molecular Therapy 10: 302–317. Carlsson T, Bjorklund T, and Kirik D (2007) Restoration of the striatal dopamine synthesis for Parkinson’s disease: Viral vector-mediated enzyme replacement strategy. Current Gene Therapy 7: 109–120. Denovan-Wright EM and Davidson BL (2006) RNAi: A potential therapy for the dominantly inherited nucleotide repeat diseases. Gene Therapy 13: 525–531. Federici T and Boulis NM (2006) Gene-based treatment of motor neuron diseases. Muscle & Nerve 33: 302–323. Jakobsson J and Lundberg C (2006) Lentiviral vectors for use in the central nervous system. Molecular Therapy 13: 484–493. Kaplitt MG, Feigin A, Tang C, et al. (2007) Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: An open label, phase I trial. Lancet 369: 2097–2105. Kaspar BK, Llado J, Sherkat N, et al. (2003) Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science 301: 839–842. Naldini L, Blomer U, Gallay P, et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263–267. Ryan DA and Federoff HJ (2007) Translational considerations for CNS gene therapy. Expert Opinion on Biological Therapy 7: 305–318. Samulski RJ (2003) AAV vectors, the future workhorse of human gene therapy. Ernst Schering Research Foundation Workshop 43: 25–40. Sands MS and Davidson BL (2006) Gene therapy for lysosomal storage diseases. Molecular Therapy 13: 839–849. St George JA (2003) Gene therapy progress and prospects: Adenoviral vectors. Gene Therapy 10: 1135–1141. Suhr ST and Gage FH (1993) Gene therapy for neurologic disease. Archives of Neurology 50: 1252–1268. Suhr ST and Gage FH (1999) Gene therapy in the central nervous system: The use of recombinant retroviruses. Archives of Neurology 56: 287–292. Tashiro A, Zhao C, and Gage FH (2006) Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo. Nature Protocols 1: 3049–3055. Tyler CM, Wuertzer CA, Bowers WJ, et al. (2006) HSV amplicons: Neuro applications. Current Gene Therapy 6: 337–350. Wu Z, Asokan A, and Samulski RJ (2006) Adeno-associated virus serotypes: Vector toolkit for human gene therapy. Molecular Therapy 14: 316–327.