Gene-Based Neuromodulation

C H A P T E R

30 Gene-Based Neuromodulation Hilarie C. Tomasiewicz, Gary Kocharian, Michael G. Kaplitt Weill Cornell Medicine, New York, NY, United States

O U T L I N E Introduction429 Gene Therapy Vectors

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Viral Vectors AAV Vectors LV Vectors HSV Vectors Adenoviral Vectors

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Control of Transgene Expression Promotor Selection Regulatable Expression Systems Additional Gene Therapy Approaches

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Human Clinical Safety and Efficacy Data for CNS Gene Therapy

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Genetic Diseases 433 Canavan Disease 434 Batten Disease (Late Infantile Neuronal Ceroid Lipofuscinosis)434 X-Linked Adrenoleukodystrophy 434 Ongoing Gene Therapy Considerations for Genetic Diseases of the CNS 434

INTRODUCTION One billion people worldwide have a neurologic disorder (World Health Organization, 2006), and despite great demand for effective therapies, treatment options for these disorders remain quite limited. Interventions to deliver genes to the nervous system that permanently restore function are a powerful alternative to existing standard pharmacologic approaches; such approaches, in which DNA or RNA is used as the therapeutic agent,

Neuromodulation, Second Edition http://dx.doi.org/10.1016/B978-0-12-805353-9.00030-9

Neurodegenerative Diseases Parkinson Disease Alzheimer Disease

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Brain Tumors

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Discussion of Future Trends and Pathways to Expanding the Knowledge Base

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Novel Disease Targets 436 Epilepsy436 Neuropsychiatric Disease 437 Novel Modulatory Gene Therapy Approaches 437 Optogenetics: A Form of Gene Therapy 437 Chemogenetics438 Magnetogenetics/Radiogenetics438 Novel Approaches for Noninvasive Gene Delivery 438 Systemic Gene Delivery 438 Magnetic Resonance Imaging–Guided Focused Ultrasound439 References439

are defined as gene therapy. Though historically fraught with controversy in the early 1990s, gene therapy has reemerged over the past decade as a safe, feasible, and targeted potential therapeutic for a range of neurologic disorders. The complexity of the nervous system has long posed a challenge to scientists and physicians looking to develop efficacious treatments for diseases affecting the central nervous system (CNS). While pharmacotherapy and deep brain stimulation (DBS) are the current mainstays for treating neurologic disease, both approaches

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have significant limitations regarding specificity, time course, and side effect profile. Ideally, next-generation therapies for neurologic disease will be noninvasive, bidirectional, spatially precise, and temporally regulatable: in the near future, gene therapy has the very real potential to meet all of these criteria. This chapter will provide a review of the relevant history and current knowledge base of gene therapy, a summary of developments over the past decade including completed and ongoing clinical trials, and a discussion of future trends and pathways toward expanding the knowledge base.

GENE THERAPY VECTORS The complexity of the nervous system and the multigenetic nature of most neurologic disorders continue to present significant challenges to the design and implementation of successful gene therapies in the clinic. The efficacy of gene therapy is dependent on effective gene transfer, and great efforts have been made since the advent of gene therapy in the mid-1980s to design vectors that efficiently deliver therapeutic genes to the nervous system to replace defective genes. The delivery of genes into cells can be accomplished by multiple methods. Nonviral gene delivery methods, such as naked nucleic acid, liposomes, and nanoparticles, while advantageous in their low cost and ability to deliver large genetic payloads, have historically resulted in low therapeutic gene expression of limited duration (Elsabahy et al., 2011), although recent advances are beginning to rectify this. The development of viral-based approaches, which exploit the innate ability of a virus to insert its DNA into host cells, however, has been the most promising method of gene delivery over the past two decades. To date, the primary viral vectors for CNS gene transfer are adeno-associated viral (AAV) and lentiviral (LV), although herpes simplex viral (HSV), and adenoviral

(AV) vectors have also been used to treat nervous system disorders and will be touched on briefly here.

VIRAL VECTORS As mentioned, the hijacking of viral biology for transgene expression rather than viral replication within host cells is key to the development of an efficacious viral vector for gene therapy. Importantly, each of these viral vectors has advantages and disadvantages for specific gene transfer needs as they vary considerably with respect to transgene insert size, duration of transgene expression, cellular target, and degree of immunogenicity. Table 30.1 depicts the characteristics of the most commonly used viral vectors. The design of any viral-based gene therapy strategy, therefore, should take into account the inherent technical strengths and limitations particular to a given viral vector.

AAV Vectors AAV vectors, derived from nonpathogenic, singlestranded DNA parvoviruses, have emerged as one of the safest and most extensively used vectors for gene delivery in both basic and clinical applications due to their ability to infect nondividing cells, high transduction efficiency, long-lasting expression from a single dose, and relatively low host immune response (Kaplitt et al., 1994). Consequently, a majority of clinical trials addressing nononcologic diseases of the CNS have used AAV vectors (LeWitt et al., 2011; McPhee et al., 2006; Worgall et al., 2008). More than 100 serotypes of AAV have been identified, each with a distinct tissue selectivity and transduction efficiency due to differences in its capsid proteins, and efforts to engineer AAV capsids to improve cell-type specificity and reduce immunogenicity are ongoing. The most commonly used AAV serotypes in the

TABLE 30.1  Viral Vectors in Clinical Use Adeno-Associated Virus

Lentivirus

Herpes Simplex Virus

Adenovirus

Genome

Single-stranded DNA

Single-stranded RNA

Double-stranded DNA

Double-stranded DNA

Transgene capacity

∼4.5 kb

∼8 kb

∼40–50 kb

∼7.5 kb

Host genome integration

No

Yes

No

No

Neuron targeting

Yes

Yes

Yes

Yes

Onset of transgene expression

Slow

Fast

Fast

Fast

Expression duration

Long

Long

Short

Short

Number of serotypes

>100

5

2

∼50

Typical gene delivery method

CNS in vivo

Ex vivo transfection of hematopoietic stem cells

CNS in vivo

CNS in vivo, brain tumors

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Control of Transgene Expression

CNS currently are 1, 2, 4, 5, 6, 8, and 9. AAV2 has been used most extensively in clinical studies, but its spread and neuronal transduction in the brain after direct injection are limited compared with the more recently characterized AAV1, AAV5, and AAV9 serotypes (Mandel and Burger, 2004). Notably, AAV9 has recently been shown to cross the blood–brain barrier (BBB) after both intravenous and intra–cerebrospinal fluid (CSF) injection, inducing widespread transgene expression throughout the CNS in the absence of direct injection (Haurigot et al., 2013). While AAV vectors remain at the forefront of gene therapy, their small packaging size (<4.7 kb) and slow onset of expression may limit the use of these vectors in settings where large or multiple genes may be required or very rapid expression is needed. Lastly, the recent development of a primate-derived AAV strain (rh10) for use in the human brain might ultimately mitigate ongoing concerns of immunogenicity as it may bypass the antihuman AAV immunity that is common in the population (Sondhi et al., 2012).

LV Vectors LV vectors, derived from the single-stranded RNA retrovirus HIV-1, have been used extensively as gene transfer tools in the CNS due to their ability to infect nondividing cells, efficiently integrate into the host genome, carry large transgenes, and allow for stable long-term transgene expression. LV vectors have been shown to transduce most cell types within the CNS, and clinical trials in which LV vectors were used to correct gene defects ex vivo have demonstrated their efficacy in a number of neurogenetic disorders (reviewed in Kantor et al., 2014). Despite their advantages, however, LV vectors are associated with a relatively high risk of insertional mutagenesis as they integrate into the host genome, thus potentially retaining the ability to induce oncogenesis. Nonintegrating LV vectors are currently in development to overcome these concerns.

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and one that has been harnessed for the ongoing development of novel therapies for chronic pain.

Adenoviral Vectors Adenoviral vectors, derived from pathogenic doublestranded DNA viruses, have historically been considered advantageous as they transduce nondividing cells, carry large transgenes, and exhibit fast transgene expression without the risk of insertional mutagenesis. However, their applications as CNS gene therapy vectors remain limited given the transient nature of their transgene expression and their high propensity to provoke significant immune responses.

CONTROL OF TRANSGENE EXPRESSION While the design of all viral-based gene therapy strategies begins with selection of an appropriate vector, optimization of the viral vector genome for robust transgene expression targeted to the specific cell type or tissue of interest remains essential. As will be reviewed in depth below, many diseases of the CNS are potentially treatable with targeted gene therapy strategies, each with significant variability in pathophysiology, CNS location, affected cell-type(s), underlying genetic defect, and requirements for therapeutic gene expression or repression. Thus, the requirements for a safe and effective gene therapy vector targeting the global genetic defects characteristic of pediatric leukodystrophies will likely differ significantly from a gene therapy targeting an adult neurodegenerative disease localized to a specific brain region. However, most neurologic disorders are likely to require tightly controlled transgene expression that is limited to specific cell populations. The modification of viral vector genomes to control transgene expression or repression in multiple domains is briefly discussed here.

Promotor Selection

HSV Vectors HSV vectors, derived from pathogenic doublestranded DNA viruses, have been widely used for CNS gene transfer in basic and clinical applications given their large transgene capacity, tropism for neurons, and lack of insertional mutagenesis. The vast majority of clinical trials using HSV vectors have focused on brain tumors (reviewed in Simonato et al., 2013) and non-CNS cancers, but HSV vectors have also been trialed as gene therapy for pain (Fink et al., 2011). Uniquely, HSV vectors undergo retrograde transport from the periphery to cell bodies, a property that allows long-term transgene expression in CNS neurons without direct CNS infusion,

In addition to viral tropism for neurons, both the level of transgene expression and cell type–specific targeting can be directed by cis-acting elements contained in the vector genome, including the selection of 5′-untranslated region (UTR), 3′-UTR, enhancer, promoter, and polyadenylation signal (Simonato et al., 2013). Of these elements, the use of cell type–specific promoters greatly enhances cellular targeting as these binding sites for specific transcription factors restrict transgene expression to genetically defined cell populations. The arsenal of cell-, tissue-, and disease-specific promoters has expanded considerably in recent years, reflecting ongoing efforts to improve the strength, duration, and

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selectivity of transgene expression. For example, human synapsin (hSyn), human thymocyte antigen (hThy1), and neuron specific enolase (NSE) are selective for neurons and glial fibrillary acidic protein (GFAP) for astrocytes; calmodulin kinase II alpha (CamK11a) selects for excitatory neurons and fugu somatostatin (fSST) for inhibitory neurons (Nathanson et al., 2009). Although full specificity of most promoters often requires far larger DNA fragments than can be accommodated by even the largest viral vectors, improved understanding of regulatory elements and combining these into synthetic promoters should help overcome this issue and provide an expanding toolkit of regulators to improve specificity of CNS gene therapy.

Regulatable Expression Systems The ability to control the timing of transgene expression can be achieved by using a ligand-inducible promotor, which allows pharmacologic control of transgene expression following viral vector administration in vivo. For example, many preclinical studies have used the tetracycline (Tet) on/off system whereby viral-mediated transgene expression is dependent on either the presence or absence of tetracycline and thus can be switched on or off with the oral administration of doxycycline. Other examples of ligand-inducible systems are reviewed in Zoltick and Wilson (2001). Several systems have also been developed to ensure that viral-mediated transgene expression can be exquisitely restricted to genetically defined cell populations, including specific neuronal subpopulations. Cre-lox neurogenetics has emerged as one experimental platform that allows for strong transgene expression in any population of cells that coexpress the DNA recombinase Cre. In this approach, a transgene can be inserted into the viral vector genome such that it can be transcribed only after inversion by Cre in vivo (reviewed in Smedemark-Margulies and Trapani, 2013). The specificity of transgene expression can then come from the targeted expression of Cre in driver mouse lines in which Cre is either expressed in a specific cell type or the expression of Cre is induced by a specific external stimulus (Fenno et al., 2011), providing a means to control the precise location of transgene expression in the CNS. A related approach involves use of a tamoxifeninducible form of Cre (Danielian et al., 1998) to regulate transgene expression. Finally, several newer strategies have been developed allowing for both temporal and spatial regulation of transgene expression and are discussed at the end of this chapter. To date, however, no strategy designed to achieve regulation of transgene expression has been successfully translated to humans, although a Phase I clinical trial for glioblastoma multiforme (GBM) using a multiple AV vector-based “Tet on” approach is being planned based on recent preclinical data (VanderVeen et al., 2016).

Additional Gene Therapy Approaches Currently, gene therapy approaches are primarily focused on delivering a gene that causes a needed protein to be expressed; however, more recently, increased understanding of nuclease function has led to more direct DNA editing, using techniques such as RNA interference (RNAi) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPRassociated protein 9), both of which can be delivered to the CNS via viral-based methods. In the context of gene therapy, RNAi provides a mechanism for specific gene silencing via engineered short hairpin RNAs (shRNAs) and microRNAs (miRNAs), molecules that target specific gene sequences, act as posttranscriptional regulators, and can be used in conjunction with viral-mediated approaches. Briefly, shRNAs and miRNAs recognize target mRNAs and induce transcriptional repression or mRNA cleavage, depending on their partial or complete sequence complementarity, respectively (Tabara et al., 1999). The most recent addition to our toolbox for genome editing is the rapidly developing CRISPR/Cas9 system. Originally discovered in bacteria, CRISPR/Cas9 requires two molecules, a guide RNA (gRNA) complementary to a given target gene sequence and a nuclease called Cas9 (Mali et al., 2013). The gRNA recognizes its complementary DNA sequence in the genome and Cas9, depending on the enzymatic variant, creates a single- or double-strand break in the DNA. Single-strand breaks can trigger homology directed repair (HDR), allowing for insertion of new genes into the genome and providing a mechanism for genetic correction of DNA mutations. Alternatively, double-stranded DNA breaks trigger nonhomologous end joining (NHEJ), resulting in targeted gene disruption and inactivation of the target gene. In the context of gene therapy, CRISPR/Cas9 can theoretically be used as a therapeutic tool to correct the causative gene mutations in monogenic recessive disorders or to inactivate the mutated allele in dominantnegative disorders. For example, it was recently shown that CRISPR/Cas9-mediated genome editing partially restored dystrophin expression in a mouse model of muscular dystrophy (Long et al., 2016).

HUMAN CLINICAL SAFETY AND EFFICACY DATA FOR CNS GENE THERAPY To date, more than 1850 gene therapy clinical trials have been completed, are ongoing, or have been approved worldwide (Ginn et al., 2013). Although overlap in gene therapy approach exists for certain CNS diseases (e.g., Parkinson disease), retroviral vectors have generally been used for ex vivo gene transfer while AAV

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Genetic Diseases

TABLE 30.2 Representative Gene Therapy Clinical Trials for Neurologic Disease Disease

Phase

Viral Vector

Transgene

Target/Route of Administration

I/II

AAV2

ASPA

Intraparenchymal injection to six brain sites

I

AAV2

hCLN2

Intraparenchymal injection to 12 brain sites

I

LV

ABCD1

Ex vivo transfection of hematopoietic stem cells

Kaplitt et al. (2007)

I

AAV2

GAD

Stereotactic unilateral injection into subthalamic nucleus

LeWitt et al. (2011)

II

AAV2

GAD

Stereotactic bilateral injection into subthalamic nucleus

Marks et al. (2008)

I

AAV2

Neurturin

Stereotactic bilateral injection into putamen

Marks et al. (2010)

II

AAV2

Neurturin

Bartus et al. (2013)

I

AAV2

Neurturin

Warren Olanow et al. (2015)

II

AAV2

Neurturin

Christine et al. (2009)

I

AAV2

AADC

Stereotactic bilateral injection into putamen

Muramatsu et al. (2010)

I

AAV2

AADC

Stereotactic bilateral injection into putamen

Palfi et al. (2014)

I/II

LV

AADC/TH/GTP cyclohydrase I

Stereotactic bilateral injection into putamen

I

AAV2

NGF

Stereotactic bilateral injection into nucleus of Meynert

Rainov (2000)

I–III

HSV1

TK

Resection cavity

Westphal et al. (2013)

I–III

AV

TK

Resection cavity

Lang et al. (2003)

I

AV

p53

Intratumoral

Chiocca et al. (2008)

I

AV

IFN-β

Intratumoral

CANAVAN DISEASE Leone et al. (2000) Janson et al. (2002) BATTEN DISEASE Worgall et al. (2008)

X-LINKED ADRENOLEUKODYSTROPHY Cartier et al. (2009) PARKINSON DISEASE

Stereotactic bilateral injection into putamen and substantia nigra

ALZHEIMER DISEASE Rafii et al. (2014) BRAIN TUMORS

AADC, aromatic amino acid decarboxylase; AAV2, adeno-associated virus serotype 2; ABCD1, gene encoding ALD (adenosine triphosphate-binding cassette transporter); ASPA, aspartoacyclase; AV, adenovirus; GAD, glutamic acid decarboxylase; GTP, guanosine triphosphate; hCLN2, human ceroid lipofuscinosis, neuronal 2; HSV1, herpes simplex virus type 1; IFN-β, interferon-β; LV, lentivirus; NAA, N-acetyl-aspartate; NGF, nerve growth factor; TH, tyrosine hydroxylase; TK, thymidine kinase.

vectors have been extensively used for in vivo gene transfer. Ex vivo refers to the genetic augmentation of cells outside the body followed by reintroduction of genetically modified cells into the patient (analogous to a bone marrow transplant), an approach that is distinct from in vivo gene transfer, where the viral vector is administered directly to the patient. Historically, ex vivo gene transfer has been used to treat the CNS in several ways: (1) hematopoietic stem cells differentiate into microglia that migrate through the brain and deliver the missing gene product to neural cells, (2) hematopoietic stem cells secrete the absent gene product into the intravascular compartment, which crosses the BBB into the brain, or (3) the immune system is corrected, profiting the nervous system. Of clinical trials focused on

neurologic disorders, the vast majority have used AAV in vivo as gene therapy vector (Gray et al., 2010). A selection of representative clinical trials of gene therapy for neurologic disease is shown in Table 30.2.

GENETIC DISEASES In the earliest days of gene therapy, the most obvious initial application was to treat inherited monogenetic diseases by transferring the functioning gene into dividing stem cells to ensure the permanence of the correction. In 2000, the first report of successful ex vivo treatment of a genetic disease, X-linked severe combined immunodeficiency, was published (Cavazzana-Calvo et al., 2000).

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Although a majority of the 20 infants treated in this trial underwent full immunologic reconstitution, five infants developed a T-cell leukemia that was the direct result of insertional mutagenesis caused by the retroviral gene transfer vector used. Though the trial was put on hold, a worldwide effort was undertaken to develop vectors with improved safety profiles, allowing further clinical trials to be initiated, including those for genetic diseases affecting the CNS. Since then, three Phase I clinical trials of gene therapy for genetic diseases of the CNS have been completed to date.

Canavan Disease Gene therapy for Canavan disease (CD) is historically significant as it represents the first clinical use of gene therapy for a neurogenetic disease. CD is a monogenetic, autosomal recessive leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and accumulation of N-acetyl-aspartate (NAA) in the brain, resulting in global spongiform degeneration of white matter and severe impairment of psychomotor development. The first Phase I clinical trial for CD involved direct intraventricular delivery of nonviral human ASPA gene therapy in 16 patients (Leone et al., 2000); overall improvements in primary outcome measures were negligible, however, prompting a second Phase I/II clinical trial using AAV-mediated gene transfer (AAV2-ASPA). In this trial, 13 patients underwent intraparenchymal delivery of AAV2-ASPA at six brain sites (Janson et al., 2002); although brain NAA levels were decreased up to 2 years and no deaths were reported at up to 10 years after gene therapy, 30% of patients exhibited immune responses (Leone et al., 2012; McPhee et al., 2006). The modest treatment effects observed in this study are likely due to very limited intraparenchymal spread of AAV2.

Batten Disease (Late Infantile Neuronal Ceroid Lipofuscinosis) Late infantile neuronal ceroid lipofuscinosis (LINCL), or Batten disease, is a monogenetic, autosomal recessive, lysosomal storage disease caused by mutations in the CLN2 gene, leading to accumulation of lipofuscin in neurons and resulting in progressive neurologic decline, psychomotor impairment, and seizures. In a Phase I clinical trial designed to assess the effects of AAV-mediated human CLN2 gene transfer on neurologic decline, 10 patients underwent image-guided intraparenchymal infusion of AAV2CUh-CLN2 to 12 locations in the brain (six burr holes with vector deposition at two depths per burr hole) (Worgall et al., 2008). While this trial suggested both a slowing in the progression of LINCL in treated patients as measured by neurologic rating scales

and no evidence of CNS inflammation, 40% of patients developed mild immune responses. As with CD, the major challenge for successful LINCL gene therapy is the requirement for wide distribution of the therapy within the brain. Although this study attempted to maximize vector spread on the basis of safety constraints limiting the volume that could be administered, the goal of spreading the vector diffusely throughout the brain will likely come from the development of new vectors that distribute more widely through the CNS (Souweidane et al., 2010). To address the limited spread of AAV2 vectors, the rh10 AAV capsid was identified as having significantly better brain distribution and expression as well as potentially less immunogenicity in rodents and nonhuman primates (Sondhi et al., 2012), and a second Phase I clinical trial for LINCL using AAVrh10 is currently under way (NCT01161576, NCT01414985).

X-Linked Adrenoleukodystrophy A Phase I clinical trial of ex vivo lentiviral gene therapy for X-linked adrenoleukodystrophy (X-ALD), a severe progressive cerebral demyelinating disease caused by deficiency of the ABCD1 gene, was the first to demonstrate safe and efficacious treatment of a neurogenetic disease by a non–AAV-mediated approach. In this trial, the hematopoietic stem cells from two patients were transfected ex vivo with an LV vector containing the ABCD1 gene and subsequently reinfused into each patient. In both patients, cerebral demyelination was ultimately arrested, and normal cognitive and motor function persisted for at least 6 years (Cartier et al., 2009, 2012). The effects of treatment were thought to be mediated by gene-corrected monocytes migrating into the CNS to replace defective brain microglia. Although studies with larger cohorts are needed, this trial was the first to demonstrate efficacious treatment of CNS disease by a lentiviral gene therapy.

Ongoing Gene Therapy Considerations for Genetic Diseases of the CNS In addition to CD, LINCL, and X-ALD, a large number of other genetic diseases affect the CNS, many of which are also characterized by pathologic neurodegeneration that is typically widely distributed throughout the brain. As mentioned, global widespread delivery of therapeutic genes remains a significant challenge to the treatment of genetic diseases affecting the CNS. Accumulating data is providing evidence that alternative AAV serotypes, such as 1, 5, 8, 9, or rh10, provide higher transgene levels and wider distribution in the brain than AAV2 (Kantor et al., 2014). While new clinical trials are under way examining how AAV serotype mediates spread of the viral vector once delivered to the brain, additional

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Neurodegenerative Diseases

important considerations concern method of viral delivery. On the basis of studies in rodents and nonhuman primates, various strategies for widespread transgene expression have shown potential for human applications, including distal transport of the viral vector via neural pathways (Cearley and Wolfe, 2007), endovascular approaches (Foust et al., 2009; Gray et al., 2011), and intra-CSF injection (Liu et al., 2005). Lastly, viral vector immunogenicity may also ultimately be overcome by further development of nonhuman viral vectors.

NEURODEGENERATIVE DISEASES While early trials for CD and LINCL used the best vector technology available at the time for global dispersion of genes to the CNS, these treatments were largely ineffective in terms of reversing disease phenotype for the reasons discussed above. These early trials, however, paved the way for AAV2 to be used to focally target other neurodegenerative disorders, including Parkinson disease (PD), Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and Huntington disease (HD), disorders that, with the exception of HD, overwhelmingly affect the aging population. The following discussion of human safety and efficacy data will focus primarily on clinical trials of gene therapy for PD, although substantial progress has been made in preclinical studies towards gene therapy for ALS and HD. Taken together, the results from human clinical trials support the overall safety of gene-based therapy approaches for the treatment of neurologic disorders.

Parkinson Disease The second most common age-related neurodegenerative disease, PD is the most frequent neurologic disorder targeted in clinical gene therapy trials to date. PD is characterized by the progressive loss of dopaminergic cells in the substantia nigra pars compacta (SNpc) and results in loss of dopamine in the striatonigral pathway relative to the striatopallidal pathway, leading to the classic symptom triad of tremor, rigidity, and bradykinesia as well as cognitive impairments that can progress to frank dementia. Since PD was first described, the mainstay of treatment involved pharmacologic replacement of the dopamine precursor levodopa; however, as the disease progresses, chronic levodopa therapy invariably becomes ineffective at symptom control and is associated with dyskinesia. While DBS continues to represent a highly effective therapy for PD, there is clear need for more targeted treatments. Given the discrete pathophysiology of PD, three main strategies have been employed in PD gene therapy clinical trials to date: (1) induction of dopamine production, (2) inhibition of the subthalamic

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nucleus (STN) through enhanced GABAergic signaling, and (3) protection of dopaminergic neurons in the SNpc (reviewed in Simonato et al., 2013), all of which were designed on the basis of extensive preclinical data demonstrating efficacy in animal models. The most straightforward approach to achieve a therapeutic level of dopamine is by targeted overexpression of enzymes and cofactors required to produce dopamine from tyrosine. In the first Phase I clinical trial to use a lentiviral vector (equine infectious anemia virus [EIAV]) to deliver three genes necessary for dopamine biosynthesis (tyrosine hydroxylase, GTP cyclohydrase I, and AADC) to the bilateral putamen of PD patients, treated patients demonstrated significant improvement in motor function up to 1 year with evidence of clinical benefit up to 4 years (Palfi et al., 2014), however, the magnitude of effects were within the placebo range reported in several other PD gene therapy trials discussed below (Christine et al., 2009; Marks et al., 2008). In a related strategy, two separate Phase I clinical trials of AAV2-mediated delivery of AADC to the bilateral putamen showed 30%–56% increases in dopamine activity as measured by PET, but both trials showed only modest improvements in patients’ motor scores (Christine et al., 2009; Muramatsu et al., 2010). A third Phase I clinical trial of AAV-AADC using higher vector dosage is currently ongoing (NCT01973543). A second strategy to PD gene therapy has been centered on altering the balance of neuronal activity in the basal ganglia specifically by reducing STN activity through overexpression of glutamate decarboxylase (GAD), the enzyme that converts glutamate to GABA (Luo et al., 2002). In a Phase I trial using an AAV2 vector encoding GAD injected unilaterally into the STN, treated patients had significantly improved Unified Parkinson Disease Rating Scale (UPDRS) scores at up to 1 year as well as corresponding improvements in thalamic metabolism on the treated side (Kaplitt et al., 2007). Moreover, patient immune response to AAV2GAD was minimal. Given these safety and tolerability findings, a Phase II trial of bilateral AAV2-GAD into the STN was initiated; treated patients again exhibited significant motor improvements from baseline at 6 months (LeWitt et al., 2011); this trial was the first to demonstrate efficacy of gene therapy for a neurodegenerative disease in a double-blind, randomized, sham-surgery controlled trial. The third approach to PD gene therapy has focused on neuroprotection, namely through overexpression of neurotrophic factors (e.g., neurturin, glial derived neurotrophic factor [GDNF]) that may prevent degeneration of dopaminergic neurons in the SNpc. In a Phase I trial, bilateral putaminal injection of AAV2-Neurturin significantly increased UPDRS motor scores at 1 year (Marks et al., 2008), however, no significant effect was detected at

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1 year in the subsequent Phase II trial (Marks et al., 2010). A second Phase I trial was initiated to test the safety of bilaterally administered AAV2-Neurturin to the putamen plus substantia nigra in an effort to account for deficiencies in axonal transport that may limit the effectiveness of neurotrophic factors (Bartus et al., 2013), however, the Phase II trial that followed showed no significant benefit of AAV2-Neurturin compared with sham-surgery controls (Warren Olanow et al., 2015). Currently, a Phase I trial of AAV2-GDNF bilaterally delivered to the putamen via convection-enhanced delivery (CED) is ongoing (NCT01621581).

Alzheimer Disease AD, the most common age-related neurodegenerative disease, continues to represent a significant and unmet medical need. Existing therapies do not slow the progressive degeneration of forebrain cholinergic neurons characteristic of AD, and cholinesterase inhibitors, the current mainstay of treatment, continue to be associated with dose-limiting toxicity. Based on preclinical studies demonstrating a role for nerve growth factor (NGF) in restoring cholinergic neuron function, a small Phase I trial assessed the safety and initial efficacy of bilaterally injected AAV2-NGF into the nucleus basalis of Meynert. Treated patients showed no evidence of accelerated decline, and autopsy tissue confirmed NGF expression persisted up to 4 years (Rafii et al., 2014).

BRAIN TUMORS The vast majority of clinical trials in gene therapy have been aimed at the treatment of cancers (Ginn et al., 2013), and a considerable amount of work has been done using a variety of viral-based approaches to treat malignant brain tumors, namely GBM. The development of gene therapy approaches for GBM stemmed from the theory that viral vectors injected into the brain may target residual tumor cells not killed by radiation and chemotherapy, the current mainstays of treatment that, together with maximal surgical resection, have increased median survival of patients from 6–9 months to 18–21 months (Grossman et al., 2010). Initial Phase I-III clinical trials used nonreplicating retroviral vectors encoding an HSV thymidine kinase (TK) gene, which sensitizes cells to ganciclovir, however, no improvements in patient survival were demonstrated (Rainov, 2000). Subsequent Phase I-III clinical trials using adenoviral vectors containing TK also ultimately failed to show a significant effect on overall survival (Westphal et al., 2013). Other adenoviral-mediated therapeutic approaches have targeted p53 and interferon-β but failed to increase survival (Chiocca et al., 2008; Lang et al., 2003). Additional clinical

trials of both nonreplicating and replication competent viral vectors that deliver conditional cytotoxic enzymes, toxins, cytokines, and shRNAs to tumor cells, however, are currently ongoing.

DISCUSSION OF FUTURE TRENDS AND PATHWAYS TO EXPANDING THE KNOWLEDGE BASE In the context of diseases of the CNS, data from human clinical studies conducted over the past decade have demonstrated that gene therapy is a safe and viable alternative treatment strategy for many neurological disorders for which currently available treatments are suboptimal. Despite the significant advances that have been made to date, however, the field of gene therapy is still in a period of relative infancy, as no US Food and Drug Administration (FDA)-approved viral gene therapies are commercially available in the United States as of 2016 (http://www.fda.gov/BiologicsBloodVaccines/ CellularGeneTherapyProducts/ucm2005904.htm). Gene therapy strategies, however, are continuing to advance rapidly. As viral vector technology and genome design evolve, methods to better control the spatial and temporal control of transgene expression are developed, and routes of administration broaden, it is almost certain that global CNS gene transfer will become part of the fabric of human medicine in the very near future. The following discussion of future trends and pathways towards expanding the gene therapy knowledge base will focus on novel disease targets in the CNS, modulatory gene therapy approaches for precise transgene control, and emerging strategies for noninvasive gene delivery.

NOVEL DISEASE TARGETS Although most current clinical trials using viral gene therapy have focused on the neurodegenerative disorders discussed in the previous section, such human trials prove the feasibility of targeted gene delivery to alter specific networks in the brain and illustrate that effective gene therapy requires defined molecular targets, known neural circuitry, and well-defined anatomic location. Thus, as the underlying mechanisms of other diseases affecting the CNS are elucidated, disorders such as epilepsy and neuropsychiatric disease are representative of new targets for which gene therapy strategies may prove efficacious.

Epilepsy Epilepsy describes a group of disorders characterized by a persistent increase in neuronal excitability that results in seizures of multiple types. Epilepsies

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Novel Modulatory Gene Therapy Approaches

associated with generalized seizures are often caused by a genetic defect, whereas those associated with focal seizures typically result from an underlying lesion in a specific brain region. It has been proposed that lesional epilepsies may be amenable to gene therapy as a discrete brain area is involved and a causal event is typically identified, which provides a therapeutic window for the prevention of additional seizures (Simonato et al., 2013), however, as global delivery methods improve, treatment of genetic seizures may also become possible. In animal models of epilepsy, viral-based gene therapy designed to increase GABA or decrease glutamate receptor function produced antiseizure effects (Haberman et al., 2002; Raol et al., 2006), but these effects were dependent on which cell population expressed the transgene: only selective inhibition of excitatory cells produced antiseizure effects. More recently, it was shown that AAV-mediated expression of the inhibitory neuropeptide Y had strong antiseizure effects independent of cell population (Noe et al., 2010).

Neuropsychiatric Disease Despite continuous research and advances in pharmacology for various neuropsychiatric disorders, significant numbers of patients remain unresponsive to current approaches. As most neuropsychiatric disorders are multifactorial, with contributions from multiple susceptibility genes, the neuroanatomic substrates and circuits of disorders such as depression and addiction remain poorly understood, which currently limits the use of targeted gene therapy strategies to treat these polygenetic disorders. For example, while deletion or overexpression of a gene of interest may provide information regarding the role of that gene in a given behavior in an animal model of psychiatric illness, identification of one or more specific brain targets mediating the effects of that gene on the behavior is critical to considering that gene as a candidate for gene therapy (Gelfand and Kaplitt, 2013). Indeed, a majority of preclinical psychiatric research is currently directed towards elucidating both the genes and the specific brain targets mediating behavior in animal models of psychiatric disorders, likely allowing for the development of targeted gene therapies in the near future.

NOVEL MODULATORY GENE THERAPY APPROACHES As discussed above in the previous section, the vast majority of gene therapy clinical trials for neurologic disease have involved in vivo viral-mediated transfer of genes into the brain with the goal of restoring genetic function either throughout the brain (e.g., in pediatric leukodystrophies) or in discrete brain regions particular

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to a given disease (e.g., PD), using the best viral vector technology available at the time. Such strategies have necessarily relied on our best current understanding of the neurocircuitry underlying a given neurologic disease, which also has remained quite limited. Moreover, our current best therapies for neurologic disease, namely, pharmacologic treatments and DBS, while remaining effective for some neurologic disorders, are limited in their specificity. For example, pharmacologic treatments can target specific receptors, but they lack the exquisite temporal precision that characterizes neural activity. Similarly, electrical stimulation, while temporally precise, affects all cellular elements (e.g., cells and fibers of passage) within the region of stimulation. Over the past decade, however, the emergence of optogenetic (Boyden et al., 2005) and other related technologies not only has begun to revolutionize our understanding of the functional neurocircuitry of many neurologic disorders but also is enabling the design of circuit-based therapeutics to control the activity of neurons. The eventual clinical application of such circuit-based approaches for neurologic disease will likely allow for more precise targeting of specific neural elements, reduce off-target effects seen with existing pharmacologic treatments and conventional electrical stimulation, and may ultimately, in conjunction with viral-mediated strategies, allow for targeted circuit-specific neural modulation. Several of these promising circuit-based approaches will be discussed below.

Optogenetics: A Form of Gene Therapy Like gene therapy, optogenetic techniques can provide, in specific cellular populations, either gain or loss of function, with the added advantage that these functions can be controlled with light of specific wavelengths (Deisseroth et al., 2006). In this approach, a lightsensitive ion channel, such as rhodopsin, is delivered to a specific neuronal population, and subsequent local light administration through an implanted fiberoptic device allows for precise spatial and temporal control of that neuronal population via neuronal activation or inhibition depending on the type of light-sensitive ion channel being expressed (Deisseroth, 2011). With optogenetics, it is possible to genetically target a cell population to achieve a specific physiological effect with the temporal resolution of an electrical device. The delivery of opsins can be targeted to specific neuronal subtypes or populations of neurons by using either cell-typespecific promotors or experimental systems selectively expressing the enzyme Cre recombinase. Thus, optogenetic approaches provide spatially targeted gene therapy that can be precisely temporally regulated. Optogenetic techniques have been extensively used in preclinical research settings to unravel the complex

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circuitry underlying many neurologic and neuropsychiatric diseases. For example, optogenetic strategies are widely used to model and interrogate the molecular and neurochemical basis of epilepsy (reviewed in Zhao et al., 2015). While the vast majority of optogenetic work to date has been done in animal models, therapeutic optogenetic approaches are only now beginning to be trialed clinically. Based on preclinical work in which delivery of an opsin conferred light sensitivity to retinal nerve cells and restored functional vision (Busskamp and Roska, 2011), the first human clinical trial of an optogenetic therapeutic has been initiated for patients with retinitis pigmentosa with the aim of improving or restoring vision for patients with photoreceptor degeneration (NCT02556736). Translational optogenetics, however, is still in its infancy, and for brain disease, optogenetic therapy will likely require both a viral vector to deliver the opsin gene as well as an implantable or external light-delivery device (Chow and Boyden, 2013), the latter of which would carry classic implant-related complications (i.e., risk of infection). However, several groups are working on developing noninvasive, implant-free optogenetic methods, one of which uses transdermal illumination of peripheral nerves to treat chronic pain (Iyer et al., 2014), the other which relies on an engineered opsin that can be activated deep in the brain by transcranial stimulation through the skull (Chuong et al., 2014).

Chemogenetics Despite their undeniable potential as neurologic therapeutics, optogenetic approaches have a number of caveats yet to be overcome before wide-scale translation to human medicine is possible: namely, they currently require an invasive indwelling light source as well as constant light stimulation to achieve effect, the latter of which has been shown to result in channel desensitization and tissue heating. Thus, an alternative method, chemogenetics, was developed that allows for cell type–specific pharmacologic control of neuronal electrical activity (Sternson and Roth, 2014). This approach is based on ion channels (PSAMs) engineered to selectively respond to synthetic small molecule ligands (PSEMs); PSAMs are first delivered to a genetically defined population of cells via a viral vector, allowing remote control of cation or chloride conductances in those neurons via BBB-permeable PSEMS, without concurrent activation of endogenous ion channels (Magnus et al., 2011). One consideration for translational optogenetics and chemogenetics is the timescale each allows for control over neuronal activity. Optogenetics affords millisecond precision but requires considerable levels of energy to be delivered to the brain for longer timescale perturbations, whereas chemogenetic strategies show slower onsets with longer lasting effects (Sternson and Roth, 2014).

Thus, one can envision being able to customize a particular therapeutic approach for a given neurologic disease based on both the genetic mechanism of action for altering neuronal function as well as the temporal dynamics of the relevant neural circuits involved.

Magnetogenetics/Radiogenetics Recently, a system for noninvasive, temporal activation or inhibition of in vivo neuronal activity was described that allows for bidirectional remote control of neuronal activity using radio waves or magnetic fields (Stanley et al., 2012, 2016). In this approach, a viral vector encoding a Cre-dependent GFP-tagged ferritin fusion protein tethered to the cation-conducting temperature-sensitive transient receptor potential vanilloid 1 (TRPV1) was targeted to glucose-sensing neurons in the ventromedial hypothalamus in mice expressing Cre in glucose-sensing neurons. Activation of these neurons by subsequent exposure to radiofrequency waves or a magnetic field stimulated feeding behavior, while inhibition of these neurons suppressed feeding behavior (Stanley et al., 2016). Like optogenetics, magnetogenetic approaches to neural modulation result in rapid alterations of neuronal activity, but without the need for a permanent implant, potentially providing a less invasive alternative to deep brain stimulation in the future.

NOVEL APPROACHES FOR NONINVASIVE GENE DELIVERY While means for targeted, temporally regulated neural modulation are required for both the elucidation of the underlying mechanisms of neurologic disease and the ultimate treatment of these disorders via gene therapy, an ideal therapeutic strategy would obviate the requirement for direct injection into the brain or CSF space. To this end, several noninvasive strategies that allow delivery of genes across the BBB are being investigated.

Systemic Gene Delivery Delivery of viral-based gene therapeutics via an endovascular route may be a promising alternative to existing invasive methods of in vivo gene transfer. As mentioned above, AAV9 vectors have been repeatedly shown to cross the BBB after intravenous injection and induce widespread transgene expression throughout the CNS (Haurigot et al., 2013); AAV9 vector gene therapy has successfully treated spinal muscular atrophy (Foust et al., 2010), CD (Ahmed et al., 2013), and Rett syndrome (Gadalla et al., 2013) in mice. Additionally, intra-arterial (IA) delivery of viral-based gene therapy was demonstrated to be well tolerated in a Phase II clinical trial using

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References

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AV-mediated delivery of TK in patients with recurrent GBM (Ji et al., 2016). In this approach, IA gene therapy was locally delivered to recurrent tumors by prior transient BBB disruption by IA mannitol, a delivery strategy that is increasingly used for dose intensification of chemotherapy to brain tumors in patients (Boockvar et al., 2011; Chakraborty et al., 2016).

evolving. If clinical applications providing a practical means of noninvasive delivery of gene therapy continue to be developed and trialed, such approaches could ultimately be used to treat genetic deficiencies in CNS disorders by precisely targeted and temporally regulated replacement and expression of missing or defective genes.

Magnetic Resonance Imaging–Guided Focused Ultrasound

References

In the past few years, magnetic resonance imaging (MRI)-guided focused ultrasound (FUS) has emerged as a promising new technology for efficient, nonsurgical delivery of viral-based gene therapy across the BBB. Transcranial FUS in conjunction with peripheral administration of an ultrasound contrast agent has been shown to increase the permeability of the BBB both locally and transiently (Hynynen et al., 2001). Oscillating ultrasound contrast agents (e.g., microbubbles), driven by FUS waves, temporarily “unlock” the BBB, allowing systemically administrated therapeutics to enter the brain parenchyma, while maintaining their bioactivity and selectivity. Coupled with MRI, FUS can be guided to target viral-based gene therapies to brain regions of interest. Using this approach, robust AAV9-mediated transgene expression has been focally targeted to both neurons and astrocytes in striatum and hippocampus (Thevenot et al., 2012) and to neurons and oligodendrocytes in the spinal cord (WeberAdrian et al., 2015) following peripheral administration of the AAV9 vector in rodent models. Highly efficient delivery to discreet brain targets has also been demonstrated using FUS coupled with intravenous infusion of AAV1/2 vectors. Transgene expression was noted to more than 1 year, with no evidence of neuronal loss or substantial immune/inflammatory responses, suggesting that transient BBB opening with FUS does not provide sufficient exposure to limit the safety or durability of AAV gene therapy (Stavarache et al., 2016). Neuronspecific AAV-mediated transgene expression has been demonstrated by using the neuron-specific promotor synapsin (Wang et al., 2015). FUS approaches have also been used to target nonviral transgene-containing nanoparticles to the rodent brain (Mead et al., 2016). Importantly, a recent Phase III clinical trial of MRIguided FUS for essential tremor demonstrated the feasibility and safety of FUS-based approaches in humans (Elias et al., 2016). Thus, transcranial FUS applications may ultimately fulfill a long-term goal of gene therapy: noninvasive, targeted delivery of genes to the CNS. This brief discussion of novel CNS disease targets, new modulatory gene therapy approaches, and emerging strategies of noninvasive gene delivery demonstrates the rapid pace at which the gene therapy field is

Ahmed, S.S., Li, H., Cao, C., Sikoglu, E.M., Denninger, A.R., Su, Q., Gao, G., 2013. A single intravenous rAAV injection as late as P20 achieves efficacious and sustained CNS Gene therapy in Canavan mice. Mol. Ther. 21 (12), 2136–2147. http://dx.doi.org/10.1038/mt. 2013.138. Bartus, R.T., Baumann, T.L., Siffert, J., Herzog, C.D., Alterman, R., Boulis, N., Olanow, C.W., 2013. Safety/feasibility of targeting the substantia nigra with AAV2-neurturin in Parkinson patients. Neurology 80 (18), 1698–1701. http://dx.doi.org/10.1212/WNL. 0b013e3182904faa. Boockvar, J.A., Tsiouris, A.J., Hofstetter, C.P., Kovanlikaya, I., Fralin, S., Kesavabhotla, K., Riina, H.A., 2011. Safety and maximum tolerated dose of superselective intraarterial cerebral infusion of bevacizumab after osmotic blood-brain barrier disruption for recurrent malignant glioma. Clinical article. J. Neurosurg. 114 (3), 624–632. http://dx.doi.org/10.3171/2010.9.JNS101223. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K., 2005. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8 (9), 1263–1268. http://dx.doi.org/10.1038/ nn1525. Busskamp, V., Roska, B., 2011. Optogenetic approaches to restoring visual function in retinitis pigmentosa. Curr. Opin. Neurobiol. 21 (6), 942–946. http://dx.doi.org/10.1016/j.conb.2011.06.001. Cartier, N., Hacein-Bey-Abina, S., Bartholomae, C.C., Bougneres, P., Schmidt, M., Kalle, C.V., Aubourg, P., 2012. Lentiviral hematopoietic cell gene therapy for X-linked adrenoleukodystrophy. Methods Enzymol. 507, 187–198. http://dx.doi.org/10.1016/B978-0-12386509-0.00010-7. Cartier, N., Hacein-Bey-Abina, S., Bartholomae, C.C., Veres, G., Schmidt, M., Kutschera, I., Aubourg, P., 2009. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326 (5954), 818–823. http://dx.doi.org/10.1126/ science.1171242. Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Fischer, A., 2000. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288 (5466), 669–672. Cearley, C.N., Wolfe, J.H., 2007. A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease. J. Neurosci. 27 (37), 9928–9940. http:// dx.doi.org/10.1523/JNEUROSCI.2185-07.2007. Chakraborty, S., Filippi, C.G., Wong, T., Ray, A., Fralin, S., Tsiouris, A.J., Boockvar, J.A., 2016. Superselective intraarterial cerebral infusion of cetuximab after osmotic blood/brain barrier disruption for recurrent malignant glioma: phase I study. J. Neurooncol. 128 (3), 405–415. http://dx.doi.org/10.1007/s11060-016-2099-8. Chiocca, E.A., Smith, K.M., McKinney, B., Palmer, C.A., Rosenfeld, S., Lillehei, K., Kirn, D., 2008. A phase I trial of Ad.hIFN-beta gene therapy for glioma. Mol. Ther. 16 (3), 618–626. http://dx.doi. org/10.1038/sj.mt.6300396. Chow, B.Y., Boyden, E.S., 2013. Optogenetics and translational medicine. Sci. Transl. Med. 5 (177), 177ps5. http://dx.doi.org/10.1126/ scitranslmed.3003101.

VI.  EMERGING TECHNOLOGIES AND TECHNIQUES

440

30.  GENE-BASED NEUROMODULATION

Christine, C.W., Starr, P.A., Larson, P.S., Eberling, J.L., Jagust, W.J., Hawkins, R.A., Aminoff, M.J., 2009. Safety and tolerability of putaminal AADC gene therapy for Parkinson disease. Neurology 73 (20), 1662–1669. http://dx.doi.org/10.1212/WNL.0b013e3181c29356. Chuong, A.S., Miri, M.L., Busskamp, V., Matthews, G.A., Acker, L.C., Sorensen, A.T., Boyden, E.S., 2014. Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Nat. Neurosci. 17 (8), 1123– 1129. http://dx.doi.org/10.1038/nn.3752. Danielian, P.S., Muccino, D., Rowitch, D.H., Michael, S.K., McMahon, A.P., 1998. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8 (24), 1323–1326. Deisseroth, K., 2011. Optogenetics. Nat. Methods 8 (1), 26–29. http:// dx.doi.org/10.1038/nmeth.f.324. Deisseroth, K., Feng, G., Majewska, A.K., Miesenbock, G., Ting, A., Schnitzer, M.J., 2006. Next-generation optical technologies for illuminating genetically targeted brain circuits. J. Neurosci. 26 (41), 10380–10386. http://dx.doi.org/10.1523/JNEUROSCI. 3863-06.2006. Elias, W.J., Lipsman, N., Ondo, W.G., Ghanouni, P., Kim, Y.G., Lee, W., Chang, J.W., 2016. A randomized trial of focused ultrasound thalamotomy for essential tremor. N. Engl. J. Med. 375 (8), 730–739. http://dx.doi.org/10.1056/NEJMoa1600159. Elsabahy, M., Nazarali, A., Foldvari, M., 2011. Nonviral nucleic acid delivery: key challenges and future directions. Curr. Drug Deliv. 8 (3), 235–244. Fenno, L., Yizhar, O., Deisseroth, K., 2011. The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389–412. http:// dx.doi.org/10.1146/annurev-neuro-061010-113817. Fink, D.J., Wechuck, J., Mata, M., Glorioso, J.C., Goss, J., Krisky, D., Wolfe, D., 2011. Gene therapy for pain: results of a phase I clinical trial. Ann. Neurol. 70 (2), 207–212. http://dx.doi.org/10.1002/ ana.22446. Foust, K.D., Nurre, E., Montgomery, C.L., Hernandez, A., Chan, C.M., Kaspar, B.K., 2009. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27 (1), 59–65. http://dx.doi.org/10.1038/nbt.1515. Foust, K.D., Wang, X., McGovern, V.L., Braun, L., Bevan, A.K., Haidet, A.M., Kaspar, B.K., 2010. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat. Biotechnol. 28 (3), 271–274. http://dx.doi.org/10.1038/ nbt.1610. Gadalla, K.K., Bailey, M.E., Spike, R.C., Ross, P.D., Woodard, K.T., Kalburgi, S.N., Cobb, S.R., 2013. Improved survival and reduced phenotypic severity following AAV9/MECP2 gene transfer to neonatal and juvenile male Mecp2 knockout mice. Mol. Ther. 21 (1), 18–30. http://dx.doi.org/10.1038/mt.2012.200. Gelfand, Y., Kaplitt, M.G., 2013. Gene therapy for psychiatric disorders. World Neurosurg. 80 (3–4), S32 e11–38. http://dx.doi. org/10.1016/j.wneu.2012.12.028. Ginn, S.L., Alexander, I.E., Edelstein, M.L., Abedi, M.R., Wixon, J., 2013. Gene therapy clinical trials worldwide to 2012-an update. J. Gene Med. 15 (2), 65–77. http://dx.doi.org/10.1002/jgm.2698. Gray, S.J., Matagne, V., Bachaboina, L., Yadav, S., Ojeda, S.R., Samulski, R.J., 2011. Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol. Ther. 19 (6), 1058–1069. http://dx.doi.org/10.1038/ mt.2011.72. Gray, S.J., Woodard, K.T., Samulski, R.J., 2010. Viral vectors and delivery strategies for CNS gene therapy. Ther. Deliv. 1 (4), 517–534. Grossman, S.A., Ye, X., Piantadosi, S., Desideri, S., Nabors, L.B., Rosenfeld, M., Consortium, Nabtt Cns, 2010. Survival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States. Clin. Cancer Res. 16 (8), 2443–2449. http://dx.doi.org/10.1158/1078-0432. CCR-09-3106.

Haberman, R., Criswell, H., Snowdy, S., Ming, Z., Breese, G., Samulski, R., McCown, T., 2002. Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity. Mol. Ther. 6 (4), 495–500. Haurigot, V., Marco, S., Ribera, A., Garcia, M., Ruzo, A., Villacampa, P., Bosch, F., 2013. Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy. J. Clin. Investig. http://dx.doi.org/10.1172/JCI66778. Hynynen, K., McDannold, N., Vykhodtseva, N., Jolesz, F.A., 2001. Noninvasive MR imaging-guided focal opening of the bloodbrain barrier in rabbits. Radiology 220 (3), 640–646. http://dx.doi. org/10.1148/radiol.2202001804. Iyer, S.M., Montgomery, K.L., Towne, C., Lee, S.Y., Ramakrishnan, C., Deisseroth, K., Delp, S.L., 2014. Virally mediated optogenetic excitation and inhibition of pain in freely moving nontransgenic mice. Nat. Biotechnol. 32 (3), 274–278. http://dx.doi.org/10.1038/nbt.2834. Janson, C., McPhee, S., Bilaniuk, L., Haselgrove, J., Testaiuti, M., Freese, A., Leone, P., 2002. Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum. Gene Ther. 13 (11), 1391– 1412. http://dx.doi.org/10.1089/104303402760128612. Ji, N., Weng, D., Liu, C., Gu, Z., Chen, S., Guo, Y., Li, N., 2016. Adenovirus-mediated delivery of herpes simplex virus thymidine kinase administration improves outcome of recurrent high-grade glioma. Oncotarget 7 (4), 4369–4378. http://dx.doi.org/10.18632/ oncotarget.6737. Kantor, B., McCown, T., Leone, P., Gray, S.J., 2014. Clinical applications involving CNS gene transfer. Adv. Genet. 87, 71–124. http://dx.doi. org/10.1016/B978-0-12-800149-3.00002-0. Kaplitt, M.G., Feigin, A., Tang, C., Fitzsimons, H.L., Mattis, P., Lawlor, P.A., During, M.J., 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 (9579), 2097–2105. http://dx.doi.org/10.1016/S0140-6736(07)60982-9. Kaplitt, M.G., Leone, P., Samulski, R.J., Xiao, X., Pfaff, D.W., O’Malley, K.L., During, M.J., 1994. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat. Genet. 8 (2), 148–154. http://dx.doi.org/10.1038/ ng1094-148. Lang, F.F., Bruner, J.M., Fuller, G.N., Aldape, K., Prados, M.D., Chang, S., et al., 2003. Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results. J. Clin. Oncol. 21 (13), 2508–2518. Leone, P., Janson, C.G., Bilaniuk, L., Wang, Z., Sorgi, F., Huang, L., et al., 2000. Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease. Ann. Neurol. 48 (1), 27–38. Leone, P., Shera, D., McPhee, S.W., Francis, J.S., Kolodny, E.H., Bilaniuk, L.T., et al., 2012. Long-term follow-up after gene therapy for canavan disease. Sci. Transl. Med. 4 (165), 165ra163. http://dx.doi. org/10.1126/scitranslmed.3003454. LeWitt, P.A., Rezai, A.R., Leehey, M.A., Ojemann, S.G., Flaherty, A.W., Eskandar, E.N., et al., 2011. AAV2-GAD gene therapy for advanced Parkinson’s disease: a double-blind, sham-surgery controlled, randomised trial. Lancet Neurol. 10 (4), 309–319. http://dx.doi.org/ 10.1016/S1474-4422(11)70039-4. Liu, G., Martins, I., Wemmie, J.A., Chiorini, J.A., Davidson, B.L., 2005. Functional correction of CNS phenotypes in a lysosomal storage disease model using adeno-associated virus type 4 vectors. J. Neurosci. 25 (41), 9321–9327. http://dx.doi.org/10.1523/ JNEUROSCI.2936-05.2005. Long, C., Amoasii, L., Mireault, A.A., McAnally, J.R., Li, H., SanchezOrtiz, E., Olson, E.N., 2016. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351 (6271), 400–403. http://dx.doi.org/10.1126/science.aad5725.

VI.  EMERGING TECHNOLOGIES AND TECHNIQUES

References

Luo, J., Kaplitt, M.G., Fitzsimons, H.L., Zuzga, D.S., Liu, Y., Oshinsky, M.L., During, M.J., 2002. Subthalamic GAD gene therapy in a Parkinson’s disease rat model. Science 298 (5592), 425–429. http:// dx.doi.org/10.1126/science.1074549. Magnus, C.J., Lee, P.H., Atasoy, D., Su, H.H., Looger, L.L., Sternson, S.M., 2011. Chemical and genetic engineering of selective ion channel-ligand interactions. Science 333 (6047), 1292–1296. http:// dx.doi.org/10.1126/science.1206606. Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., et al., 2013. RNA-guided human genome engineering via Cas9. Science 339 (6121), 823–826. http://dx.doi.org/10.1126/science.1232033. Mandel, R.J., Burger, C., 2004. Clinical trials in neurological disorders using AAV vectors: promises and challenges. Curr. Opin. Mol. Ther. 6 (5), 482–490. Marks Jr., W.J., Bartus, R.T., Siffert, J., Davis, C.S., Lozano, A., Boulis, N., et al., 2010. Gene delivery of AAV2-neurturin for Parkinson’s disease: a double-blind, randomised, controlled trial. Lancet Neurol. 9 (12), 1164–1172. http://dx.doi.org/10.1016/S1474-4422(10)70254-4. Marks Jr., W.J., Ostrem, J.L., Verhagen, L., Starr, P.A., Larson, P.S., Bakay, R.A., et al., 2008. Safety and tolerability of intraputaminal delivery of CERE-120 (adeno-associated virus serotype 2-neurturin) to patients with idiopathic Parkinson’s disease: an open-label, phase I trial. Lancet Neurol. 7 (5), 400–408. http://dx.doi.org/10.1016/ S1474-4422(08)70065-6. McPhee, S.W., Janson, C.G., Li, C., Samulski, R.J., Camp, A.S., Francis, J., et al., 2006. Immune responses to AAV in a phase I study for Canavan disease. J. Gene Med. 8 (5), 577–588. http://dx.doi.org/ 10.1002/jgm.885. Mead, B.P., Mastorakos, P., Suk, J.S., Klibanov, A.L., Hanes, J., Price, R.J., 2016. Targeted gene transfer to the brain via the delivery of brain-penetrating DNA nanoparticles with focused ultrasound. J. Control Release 223, 109–117. http://dx.doi.org/10.1016/j.jconrel.2015.12.034. Muramatsu, S., Fujimoto, K., Kato, S., Mizukami, H., Asari, S., Ikeguchi, K., et al., 2010. A phase I study of aromatic L-amino acid decarboxylase gene therapy for Parkinson’s disease. Mol. Ther. 18 (9), 1731–1735. http://dx.doi.org/10.1038/mt.2010.135. Nathanson, J.L., Jappelli, R., Scheeff, E.D., Manning, G., Obata, K., Brenner, S., Callaway, E.M., 2009. Short promoters in viral vectors drive selective expression in mammalian inhibitory neurons, but do not restrict activity to specific inhibitory cell-types. Front. Neural Circuits 3, 19. http://dx.doi.org/10.3389/neuro.04.019.2009. Noe, F., Vaghi, V., Balducci, C., Fitzsimons, H., Bland, R., Zardoni, D., et al., 2010. Anticonvulsant effects and behavioural outcomes of rAAV serotype 1 vector-mediated neuropeptide Y overexpression in rat hippocampus. Gene Ther. 17 (5), 643–652. http://dx.doi. org/10.1038/gt.2010.23. Palfi, S., Gurruchaga, J.M., Ralph, G.S., Lepetit, H., Lavisse, S., Buttery, P.C., et al., 2014. Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson’s disease: a dose escalation, open-label, phase 1/2 trial. Lancet 383 (9923), 1138–1146. http://dx.doi.org/10.1016/S0140-6736(13)61939-X. Rafii, M.S., Baumann, T.L., Bakay, R.A., Ostrove, J.M., Siffert, J., Fleisher, A.S., Bartus, R.T., 2014. A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer’s disease. Alzheimers Dement. 10 (5), 571–581. http://dx.doi.org/10.1016/j.jalz.2013.09.004. Rainov, N.G., 2000. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum. Gene Ther. 11 (17), 2389–2401. http://dx.doi.org/10.1089/104303400750038499. Raol, Y.H., Lund, I.V., Bandyopadhyay, S., Zhang, G., Roberts, D.S., Wolfe, J.H., Brooks-Kayal, A.R., 2006. Enhancing GABA(A) receptor alpha 1 subunit levels in hippocampal dentate gyrus inhibits epilepsy development in an animal model of temporal lobe epilepsy. J. Neurosci. 26 (44), 11342–11346. http://dx.doi.org/10.1523/ JNEUROSCI.3329-06.2006.

441

Simonato, M., Bennett, J., Boulis, N.M., Castro, M.G., Fink, D.J., Goins, W.F., et al., 2013. Progress in gene therapy for neurological disorders. Nat. Rev. Neurol. 9 (5), 277–291. http://dx.doi.org/10.1038/ nrneurol.2013.56. Smedemark-Margulies, N., Trapani, J.G., 2013. Tools, methods, and applications for optophysiology in neuroscience. Front. Mol. Neurosci. 6, 18. http://dx.doi.org/10.3389/fnmol.2013.00018. Sondhi, D., Johnson, L., Purpura, K., Monette, S., Souweidane, M.M., Kaplitt, M.G., et al., 2012. Long-term expression and safety of administration of AAVrh.10hCLN2 to the brain of rats and nonhuman primates for the treatment of late infantile neuronal ceroid lipofuscinosis. Hum. Gene Ther. Methods 23 (5), 324–335. http:// dx.doi.org/10.1089/hgtb.2012.120. Souweidane, M.M., Fraser, J.F., Arkin, L.M., Sondhi, D., Hackett, N.R., Kaminsky, S.M., et al., 2010. Gene therapy for late infantile neuronal ceroid lipofuscinosis: neurosurgical considerations. J. Neurosurg. Pediatr. 6 (2), 115–122. http://dx.doi.org/10.3171/201 0.4.PEDS09507. Stanley, S.A., Gagner, J.E., Damanpour, S., Yoshida, M., Dordick, J.S., Friedman, J.M., 2012. Radio-wave heating of iron oxide nanoparticles can regulate plasma glucose in mice. Science 336 (6081), 604– 608. http://dx.doi.org/10.1126/science.1216753. Stanley, S.A., Kelly, L., Latcha, K.N., Schmidt, S.F., Yu, X., Nectow, A.R., et al., 2016. Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism. Nature 531 (7596), 647–650. http://dx.doi.org/10.1038/nature17183. Stavarache, M.A., Petersen, N., Kaplitt, M.G., 2016. Efficiency, safety, and stability of noninvasive MRgFUS mediated gene therapy in the brain. In: Program No. 673.10. 2016 Neuroscience Meeting Planner., San Diego, CA: Society for Neuroscience, 2016 Online. Sternson, S.M., Roth, B.L., 2014. Chemogenetic tools to interrogate brain functions. Annu. Rev. Neurosci. 37, 387–407. http://dx.doi. org/10.1146/annurev-neuro-071013-014048. Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., et al., 1999. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99 (2), 123–132. Thevenot, E., Jordao, J.F., O’Reilly, M.A., Markham, K., Weng, Y.Q., Foust, K.D., et al., 2012. Targeted delivery of self-complementary adeno-associated virus serotype 9 to the brain, using magnetic resonance imaging-guided focused ultrasound. Hum. Gene Ther. 23 (11), 1144–1155. http://dx.doi.org/10.1089/hum.2012.013. VanderVeen, N., Raja, N., Yi, E., Appelman, H., Ng, P., Palmer, D., et al., 2016. Preclinical efficacy and safety profile of allometrically scaled doses of doxycycline used to turn “on” therapeutic transgene expression from high-capacity adenoviral vectors in a glioma model. Hum. Gene Ther. Methods 27 (3), 98–111. http://dx.doi. org/10.1089/hgtb.2015.168. Wang, S., Olumolade, O.O., Sun, T., Samiotaki, G., Konofagou, E.E., 2015. Noninvasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus. Gene Ther. 22 (1), 104–110. http://dx.doi.org/10.1038/gt.2014.91. Warren Olanow, C., Bartus, R.T., Baumann, T.L., Factor, S., Boulis, N., Stacy, M., et al., 2015. Gene delivery of neurturin to putamen and substantia nigra in Parkinson disease: a double-blind, randomized, controlled trial. Ann. Neurol. 78 (2), 248–257. http://dx.doi. org/10.1002/ana.24436. Weber-Adrian, D., Thevenot, E., O’Reilly, M.A., Oakden, W., Akens, M.K., Ellens, N., et al., 2015. Gene delivery to the spinal cord using MRI-guided focused ultrasound. Gene Ther. 22 (7), 568–577. http://dx.doi.org/10.1038/gt.2015.25. Westphal, M., Yla-Herttuala, S., Martin, J., Warnke, P., Menei, P., Eckland, D., et al., 2013. Adenovirus-mediated gene therapy with sitimagene ceradenovec followed by intravenous ganciclovir for patients with operable high-grade glioma (ASPECT): a randomised, open-label, phase 3 trial. Lancet Oncol. 14 (9), 823–833. http://dx.doi.org/10.1016/S1470-2045(13)70274-2.

VI.  EMERGING TECHNOLOGIES AND TECHNIQUES

442

30.  GENE-BASED NEUROMODULATION

Worgall, S., Sondhi, D., Hackett, N.R., Kosofsky, B., Kekatpure, M.V., Neyzi, N., et al., 2008. Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus expressing CLN2 cDNA. Hum. Gene Ther. 19 (5), 463–474. http://dx.doi.org/10.1089/hum.2008.022. World Health Organization, WHO, 2006. Neurological Disorders: Public Health Challenges. WHO Press, Geneva, Switzerland.

Zhao, M., Alleva, R., Ma, H., Daniel, A.G., Schwartz, T.H., 2015. Optogenetic tools for modulating and probing the epileptic network. Epilepsy Res. 116, 15–26. http://dx.doi.org/10.1016/j.eplepsyres. 2015.06.010. Zoltick, P.W., Wilson, J.M., 2001. Regulated gene expression in gene therapy. Ann. N. Y. Acad. Sci. 953, 53–63.

VI.  EMERGING TECHNOLOGIES AND TECHNIQUES