Gene Therapy in Neurological Disorders

Gene therapy has tremendous potential for the treatment of neurological disorders. There has been substantial progress in the development of gene therapy strategies for neurological disorders over the last two decades. Gene Therapy in Neurological Disorders thoroughly reviews currently available gene therapy tools and presents examples of their application in a variety of neurological diseases. The book begins with general reviews of gene therapy strategies with a focus on neurological disorders. The remainder of the chapters present approaches to specific neurological disorders. Each chapter gives an in-depth introduction to the relevant field before diving into the specific tool or application. The book aims to help investigators, students and research staff better understand the principles of gene therapy and its application in the nervous system.

• Provides background information and experimental details of gene therapy tools applied for neuroscience research and neurological disorders
• Covers a broad range of gene delivery and regulation tools, therapeutic agents, and target cells, including emerging new technologies such as CRISPR/Cas9 genome editing
• Discusses applications of gene therapy tools to neurological disorders including neurodegeneration, muscular dystrophy, trauma and chronic pain, and neoplastic diseases

• Language: English
• Publisher: Academic Press
• Release date: May 25, 2018
• ISBN: 9780128098219

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Preface

Neurological disorders are among the most common and severe threats to human health and quality of life. Age-related neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, will affect more people as the population continues to age. Treatment of neurological disorders remains very challenging, however, for many reasons, including the complexity of the nervous system, the limited regenerative capacity of nervous tissue, and the difficulty inherent in delivering conventional drug treatments to nervous tissue. The brain is protected by the blood–brain barrier, a hindrance to many conventional drugs, and methods for direct delivery of treatments to the central nervous system (CNS), specifically to the brain, are often invasive and pose unacceptable risks. Gene therapy represents a way to deliver therapeutic genes and other therapeutics into the CNS and can result in permanent or regulatable expression of gene products in specific target sites. Gene therapy has tremendous potential for the treatment of neurological disorders. There has been substantial progress in the development of gene therapy strategies for neurological disorders over the last two decades. Numerous animal models have been tested and some clinical trials have already been conducted. In the past year, clinical trials using oligonucleotides and adeno-associated viral vectors to treat infantile spinal muscular atrophy have provided dramatic evidence that gene therapy approaches can have an impact on uniformly fatal neurological disorders. Although many issues remain to be solved before gene therapy approaches are available for routine use, these approaches will get much more attention in the coming years.

We have operated a viral vectors core facility for neuroscience investigators (the Hope Center Viral Vectors Core at Washington University School of Medicine) for the past 12 years. We have worked with many neuroscience investigators on basic science and preclinical studies. Investigators must make decisions about what type of vector to use and what promoters and regulatory elements should be utilized for gene delivery. Other questions include background information about gene therapy tools and their usefulness in establishing disease animal models and potential clinical applications in the treatment of neurological disorders. Gene Therapy in Neurological Disorders provides answers to these questions by thoroughly reviewing currently available gene therapy tools and presenting examples of their application in a variety of neurological diseases.

In the first section, we present general reviews of gene therapy strategies with a focus on neurological disorders. Chapter 1, Gene Therapy Methods and Their Applications in Neurological Disorders, provides a general overview of viral and nonviral gene therapy tools commonly used in neuroscience applications. It covers the mostly used viral vectors including lentiviral, adeno-associated virus, adenovirus, and herpes simplex virus. The nonviral technologies include nanoparticle-based gene delivery systems and DNA transposones. Strategies for transgene regulation, including optogenetics and receptors exclusively activated by designer drugs, are discussed. The other chapters in this section emphasize more specific approaches, including RNA interference-based gene silencing, genomic DNA delivery vectors, and stem cell–based gene therapy. The remainder of the chapters present approaches to specific neurological disorders, including neurodegenerative disorders such as Alzheimer’s disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and cerebellar neurodegenerative disorders. Additional chapters cover approaches to genetic disorders, trauma and chronic pain, and neoplastic diseases. Readers will find a broad range of gene delivery and regulation tools, therapeutic agents, and target cells, including potential application of genome editing technology, especially the newly emerged CRISPR/Cas9 methodologies. These technologies and methods are rapidly evolving, so some chapters provide a review of current knowledge and others include more specific examples of experiments and step-by-step protocols.

The goal of this book is to help investigators, students, and research staff better understand the principles of gene therapy and its application in the nervous system. We hope the work presented by the contributors’ laboratories will inspire researchers in this field to develop more gene therapy strategies that may ultimately be translated into the clinic to treat or cure neurological disorders. We thank all our contributors for their efforts, and thank all those who continue to work to find new treatments for these devastating disorders.

Section I

Overview and Methods

Outline

Chapter 1 Gene Therapy Methods and Their Applications in Neurological Disorders

Chapter 2 Targeting Transgene and RNA Interference-Based Gene Silencing Sequences to Astrocytes Using Viral Vector-Mediated Approaches

Chapter 3 Gene Therapy Using Genomic DNA: Advances and Challenges

Chapter 4 Stem Cell-Based Gene Therapy in Neurological Disorders

Chapter 1

Gene Therapy Methods and Their Applications in Neurological Disorders

Mingjie Li and B. Joy Snider,    Washington University School of Medicine, St. Louis, MO, United States

Abstract

Gene therapy is broadly defined as using nucleic acids as a treatment for disease, including replacing defective genes with normal ones. Gene therapy approaches to diseases of the nervous system are very important as treatments for these often-devastating disorders are limited or not available at all. Gene therapies are being tested in human clinical trials and also provide powerful research tools. A better understanding of the currently available gene therapy methods, including gene delivery vehicles, expression systems, and gene regulation tools, is critical for the success of a gene therapy project and for developing new gene therapy strategies. In this chapter, we describe viral and nonviral vectors for gene delivery into the central nervous system, including advantages, shortcomings, and applications of these vectors. We also introduce tools for regulating transgene expression, including optogenetics and Designer Receptors Exclusively Activated by Designer Drugs. We will discuss strategies of gene overexpression, gene silencing, and genome editing. We also provide examples of application of each strategy in animal models or clinical trials of neurological diseases such as lysosomal storage diseases, neurodegenerative disorders, and Duchenne muscular dystrophy. Our goal is to present an overview of methods used in gene therapy so that neurological researchers can choose the appropriate tools for research projects and for development of new gene therapies.

Keywords

Gene therapy; viral vector; lentivirus; adeno-associated virus; nonviral vector; nanoparticle; promoter; DREADDs; RNA interference; genome editing

Chapter Outline

1.1 Introduction 3

1.2 Viral Vectors 4

1.2.1 Retrovirus/Lentivirus 5

1.2.2 Adenovirus 10

1.2.3 Adeno-Associated Virus 11

1.2.4 Herpes Simplex Virus 13

1.3 Nonviral Vectors 13

1.3.1 Nanoparticles 14

1.3.2 DNA Transposons 15

1.4 Regulation of Transgene Expression 15

1.4.1 Regulatable Promoters 16

1.4.2 Cre/loxP System 16

1.4.3 Optogenetics 17

1.4.4 Designer Receptors Exclusively Activated by Designer Drugs 18

1.4.5 Expression of Multiple Functional Proteins or RNAs in a Single Vector 18

1.5 Modes of Gene Therapy 19

1.5.1 Overexpression of Genes 19

1.5.2 Inhibition of Gene Expression 20

1.5.3 Genome Editing 22

1.6 Conclusions 23

References 25

1.1 Introduction

Although there has been a great progress in treating some neurological disorders and many promising treatments are being tested, effective treatments are still elusive for many disorders, including Alzheimer’s disease, Huntington’s disease (HD), muscular dystrophies, and many more. Conventional small molecule treatments have not been effective for some of these disorders for many reasons, including the complexity of the nervous system and of the disorders that affect it and the challenges inherent in getting a drug to a specific target in the central nervous system (CNS) without having off-target side effects. Gene therapy, delivery of genetic materials encoding therapeutic agents (protein, RNA, etc.) into the CNS, can result in permanent or regulatable expression of a gene product in very specific target cells, so it holds promise for the treatment of a number of neurological disorders, including those caused by genetic mutations and acquired or sporadic diseases. The methods used for gene therapy include design of therapeutic DNA or RNA constructs, generation of gene transfer vectors, delivery of genes into the target cells, and regulation of transgene expression. In this review, we introduce the common tools used to transfer genes into the CNS, including viral and nonviral vectors. We describe the main properties of each vector and discuss applications, advantages, and shortcomings of these vectors. In the section about regulation of transgene expression, in addition to regulatable promoters and Cre/loxP recombination system, we introduce optogenetic approaches and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) for spatial and temporal regulation of gene expression. We also evaluate strategies for delivery and expression of multiple transgenes in a single vector construct. Finally, we present basic models of gene therapy, covering overexpression of genes, inhibition of gene expression by RNA interference (RNAi), etc., and genome editing including the recently developed CRISPR/Cas9 technology. In this chapter, we provide many examples of how these gene therapy approaches can be used in preclinical and clinical studies of neurological disorders.

1.2 Viral Vectors

The basic idea behind gene therapy is that a new piece of genetic information can be introduced into a cell or entire organism and will cure a disease or slow down a disease process. The first step in achieving this goal is to transfer the genetic material, often a gene, into the target cells. This can be difficult in any cell type but is much more difficult in cells from the CNS, and even harder still in neurons, the cells that make up the circuits of the brain and are often affected in neurological disorders. Viral vectors are the most efficient vehicles to achieve long-term stable gene expression in the CNS. Viral vectors, as the name implies, are derived from viruses and have been developed to retain the innate property of the viruses to efficiently deliver genetic material into target cells but not to cause disease or other deleterious effects in those cells. Viral vectors have been developed from many different viruses, including those that cause serious diseases like AIDS or rabies. The vectors have been developed so that genes that are required for infection are either missing altogether or are provided in separate packaging constructs, so that there is very little chance that a wild-type or infectious virus will be created. Viral vectors are derived from viruses with RNA or DNA genomes and can be either integrating or nonintegrating vectors, meaning they integrate into the host cell’s genome or remain unintegrated, or episomal. Integrating vectors typically allow for longer term expression of the therapeutic genes in the targets cells. However, efficient gene transduction can also be achieved from viral vectors that are maintained as episomes, especially in nondividing cells like neurons. The most characterized and widely used viral vectors are derived from retrovirus/lentivirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus (HSV). These viral vectors differ in the capacity (size) of genetic material that can be inserted, in their tropism (types of cells they can transduce), in the duration of transgene expression, and in potential adverse effects (see Table 1.1).

Table 1.1

dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

aRecombinant.

bGutless.

cAmplicon.

1.2.1 Retrovirus/Lentivirus

Retroviruses are a large family of enveloped RNA viruses. The viral RNA genome is converted into a double-stranded proviral DNA by the reverse transcriptase. Retroviruses are able to efficiently integrate into the genomic DNA of target cells and transfer the genetic materials into progeny of these cells. Most gene transfer vectors are derived from gamma retrovirus (oncoretrovirus, prototype: murine leukemia virus (MLV)) and lentivirus (prototype type: HIV-1) in the Retroviridae family. MLV vectors have been extensively used for gene transfer into hematopoietic cells and ex vivo transduction of cultured cells followed by transplantation (Blaese et al., 1995; Cavazzana-Calvo et al., 2000). However, these vectors have limited applications in the CNS owing to their inability to deliver genes to nondividing cells (Lewis & Emerman, 1994; Suzuki & Craigie, 2007) such as neurons. In contrast, lentiviral vectors have evolved the ability to transduce nondividing cells (Blomer et al., 1997; Naldini, Blomer, Gage, Trono, & Verma, 1996) as well as dividing cells, allowing efficient in vivo delivery, integration, and stable expression of transgenes. Lentiviral vectors have been used extensively as gene transfer tools in the CNS as they transduce most cell types in the brain, resulting in high-level and long-term transgene expression. We will focus on lentiviral vectors here.

Like other members of the retroviral family, the lentiviral RNA genome contains three essential genes, gag, pol, and env, flanked by long terminal repeats (LTRs). The gag gene encodes structure proteins and the pol gene encodes viral enzymes for reverse transcription and integration. The env gene encodes envelope glycoproteins, which mediate virus entry into the target cell. After the viral particle has bound to the receptor at the cell surface, the virion and host membranes fuse together, and the virion core is delivered into the cytoplasm of the infected cells. The viral RNA genome is subsequently converted into a double-stranded DNA by reverse transcription. The viral DNA is associated with viral proteins in a preintegration complex and translocates into the nucleus, where the integration of provirus into the host cell genome takes place (Goff, 2013).

In addition to the three essential genes (gag, pol, and env), the most extensively studied lentivirus, HIV-1, has two regulatory genes (tat and rev) required for virus replication and four accessory genes (vif, vpr, vpu, and nef) that encode virulence factors. The very first publication describing an HIV-based lentiviral vector (Naldini, Blomer, Gallay et al., 1996) demonstrated efficient transduction of neurons in vivo. In the first-generation lentiviral vectors, all the HIV-1 proteins except the envelope protein were included in the vector packaging system. To improve safety and reduce the chance of creating a wild-type or infectious virus, newer versions of lentiviral vectors were developed. In the second generation of lentiviral vectors, all the four accessary protein genes were deleted (Zufferey, Nagy, Mandel, Naldini, & Trono, 1997). In the third generation of lentiviral vectors, a constitutive promoter replaced the HIV U3 in the 5′ LTR, making the vector genome transcription independent of Tat protein. Thus, the tat gene was eliminated from the packaging system. The rev gene is required for packaging, but in newer systems it has been removed from the packaging plasmid and supplied by a separate plasmid in trans (Dull et al., 1998). The resulting gene delivery system, which relies on four separate plasmids to produce transducing particles, offers significant advantages in biosafety. Furthermore, in self-inactivated (SIN) lentiviral vectors, the U3 region of 3′ LTR is almost completely deleted from the transfer construct. The U3 region contains the viral enhancer and promoter. Transduction of a vector deleted in the 3′ U3 results in duplication of the deletion in the 5′ LTR and the transcriptional inactivation of both LTRs (Zufferey et al., 1998) (Yu et al., 1986). Fig. 1.1 shows the third-generation packaging system for producing SIN lentiviral vectors.

Figure 1.1 The third-generation packaging system for producing SIN lentiviral vectors.

The HIV-1 provirus is shown on the top. Four separate plasmids are required to generate a lentiviral vector. A transfer vector (pRRLsinPGK-GFPppt is used as an example) contains a hybrid 5′ LTR in which the U3 region is replaced with a promoter from Rous sarcoma virus (RSV), the packaging signal (ψ), the Rev response element (RRE) sequence, the central polypurine tract (cPPT) of HIV, a gene of interest along with its promoter, and the 3′ LTR in which the cis-regulatory sequences are almost completely removed from the U3 region. pMD-Lg/pRRE provides gag and pol gene products. pRSV-Rev encodes Rev, which binds to the RRE for efficient RNA export from nucleus. pCMV-G contains the vesicular stomatitis VSV-G that replaces HIV-1 Env; pA, polyadenylation signal.

Lentiviral vectors are commonly produced by cotransfection of 293T cells with a transfer plasmid and other helper plasmids encoding the structural and functional proteins required for vector packaging (Cockrell & Kafri, 2007; Li, Husic, Lin, & Snider, 2012). Stable packaging cell lines that express these needed helpers have also been developed (Haack et al., 2004; Xu, Ma, McCown, Verma, & Kafri, 2001). Because lentiviral and other retroviral vectors integrate into the host genome, there is a risk of insertional mutagenesis for retroviruses, meaning that integration of the vector could change the host genes so that cells begin to replicate and undergo oncogenic transformation; this has been documented in animal models (Seggewiss et al., 2006) and in a clinical trial for X-linked severe combined immunodeficiency (Hacein-Bey-Abina et al., 2003) when gamma retrovirus–based vectors were used. This risk of insertional mutagenesis has led to the development of nonintegrating vectors. Nonintegrating lentiviral vectors are especially suitable for gene expression in nondividing cells because episomal DNA constitutes the vast majority of viral genomes (Teo et al., 1997; Chun et al., 1997) and is exceptionally stable in nondividing cells. Integration defective lentiviral vectors can be generated by mutations in the integrase coding sequence. An integrase-deficient genome is capable of being efficiently transcribed, although the levels of protein expression are significantly lower than that achieved by conventional vectors (Bayer et al., 2008; Kantor, Ma, Webster-Cyriaque, Monahan, & Kafri, 2009). Nonintegrating lentiviral vectors can support sustained transgene expression in mouse brain (Philippe et al., 2006) and were able to improve visual function after ocular delivery of viral vector in a mouse model of retinal dystrophy (Yanez-Munoz et al., 2006).

Lentiviral vectors can be produced with different envelope glycoproteins than those from the native virus (pseudotyping). The most widely used envelope glycoprotein is from vesicular stomatitis virus (VSV-G). The receptors for VSV-G (low-density lipoprotein receptors) (Finkelshtein, Werman, Novick, Barak, & Rubinstein, 2013) are ubiquitously expressed on cell membranes, including expression in both neurons and glia. Besides the increased tropism, VSV-G is structurally more stable, so that the virus can be highly concentrated by ultracentrifugation (Burns, Friedmann, Driever, Burrascano, & Yee, 1993; Naldini, Blomer, Gallay et al., 1996). In addition to VSV-G, other glycoproteins that are used to pseudotype lentiviral vectors for gene delivery into the CNS include rabies virus glycoprotein (RV-G). These glycoproteins allow for retrograde axonal transport and transduction of neurons at a distance from the site of entry. For example, when RV-G-pseudotyped equine infectious anemia virus was injected into a muscle in the leg, transgene expression can be detected in spinal cord motor neurons; when the vector was injected into striatum, expression was detected in projecting neurons in the substantia nigra (Mazarakis et al., 2001). In HIV-based lentiviral vectors, RV-G has been shown to enhance the efficiency of gene transfer through retrograde axonal transport in both mouse and monkey brains (Kato et al., 2007). Glycoproteins from Mokola virus, lymphocytic choriomeningitis virus (LCMV), Ross River virus, and MLV have also been used to pseudotype lentiviral vectors for transducing CNS cells in vitro and in vivo (Table 1.2).

Table 1.2

A more widely used strategy to achieve targeted transduction in the CNS is by using lentiviral vectors that drive transgene expression using promoters that selectively express genes only in specific target cells. When maximal expression of a transgene is required and discrimination of cell types is not critical, a ubiquitous promoter may be preferred. CMV (cytomegalovirus) (Blomer et al., 1997; Jakobsson, Ericson, Jansson, Bjork, & Lundberg, 2003), CAG (a hybrid of CMV enhancer and chicken β-actin promoter) (Jakobsson et al., 2003), PGK (phosphoglycerate kinase) (Deglon et al., 2000; Delzor et al., 2012), and EF-1α (elongation factor-1 alpha) (Jakobsson et al., 2003) promoters have been shown to support gene expression in the brain. Although the CMV promoter is one of the strongest promoters in most mammalian cells, it is prone to transcriptional silencing associated with DNA methylation, making transgene expression decrease over time (Brooks et al., 2004). Mutation of some CpG sites may stabilize CMV-driven gene expression (Moritz, Becker, & Gopfert, 2015). In many applications, cell type-specific promoters are more desirable. For example, the expression of transgenes exclusively in astrocytes would allow for local delivery of secreted trophic factor, circumventing the anterograde transport that may induce adverse effects. In addition to ubiquitous promoters, a variety of cell type-specific promoters are available (Table 1.3). Both synapsin 1 and NSE (neuron-specific enolase) promoters show high specificity for neuronal expression in the brain (Delzor et al., 2012; Hioki et al., 2007; Jakobsson et al., 2003). GFAP promoter (glial fibrillary acidic protein) is astrocyte specific as demonstrated both in vivo (Jakobsson et al., 2003) and in vitro (Li et al., 2012). MBP (myelin basic protein) promoter drives gene expression exclusively in oligodendrocytes (McIver et al., 2005). Many endogenous neuron subtype-specific promoters are too large to put into viral vectors. The truncated versions of the vectors may be too weak to drive gene expression or lose cell-type specificity (Nathanson et al., 2009). Screening new promoters or developing synthetic promoters are required in the future.

Table 1.3

1.2.2 Adenovirus

Adenovirus (Ad) contains a double-stranded DNA genome of about 36 kb that is packaged in a nonenveloped icosahedral protein capsid (Campos & Barry, 2007). The viral DNA does not normally integrate into the host genome, so that the risk of insertional mutagenesis is minimal. The large packaging capacity, the ability to propagate high-titer viral stock, and a broad range of infectivity of cell types (including nondividing cells) make adenovirus an attractive choice for vector development (Lentz, Gray, & Samulski, 2012).

Most adenoviral vectors are derived from human Ad serotype 5, although other serotypes and nonhuman adenovirus have also been used. Recombinant Ad vectors are designed by replacing the viral sequence that required for replication with transgene expression cassettes. Wild-type adenovirus can only accommodate 2 kb of foreign DNA. By deleting the E1 region, the cloning capacity can be increased to about 5 kb (Bett, Haddara, Prevec, & Graham, 1994; Danthinne & Imperiale, 2000; Ng et al., 1999). Because E1 is essential for vector production, the vector must be propagated in E1 expressing cell line, such as HEK293. The deletion of the E1 region not only increases the cloning capacity but also renders the vector replication deficient, which is important in respect to safety for gene therapy. The E3 region is not essential for vector production. Deletion of both E1 and E3 early regions from Ad (e.g., AdEasy system) allows recombinant vectors with inserts up to 8 kb in size (He et al., 1998; Luo et al., 2007).

Helper-dependent adenoviral vectors (also referred to as gutless or gutted vectors) are vectors in which all adenovirus genomic sequences have been deleted except for the non-coding inverted terminal repeats and packaging signal, reducing host cell immunoresponse and freeing more space for transgenes (Amalfitano, 1999; Hartigan-O'Connor, Barjot, Salvatori, & Chamberlain, 2002). These vectors have a transgene capacity of approximately 36 kb. However, they still induce the innate immune response in vivo but do have attenuated adaptive immune responses (Muruve et al., 2004). Helper-dependent vectors have been shown to transduce both neuronal and glial cells in vivo (Candolfi et al., 2007). Ad5 vector with native tropism provided limited gene transfer to the brain, with the majority of gene expression in glia rather than neurons. Canine adenovirus type 2 (CAdV2 or CAV-2) vectors preferentially transduce neurons and undergo retrograde axonal transport efficiently (Soudais, Laplace-Builhe, Kissa, & Kremer, 2001). CAV-2 vectors induce a low level of immunoresponse compared with human adenovirus serotype 5 vectors (Perreau et al., 2007). A tropism-modified Ad5 vector containing the fiber knob domain from canine Ad serotype 2 has been developed. The resulting Ad5-CGW-CK2 vector can selectively transduce neurons after injection into mouse brain (Lewis, Glasgow, Harms, Standaert, & Curiel, 2014). Adenoviral vectors exhibit some features of an ideal vector but still have to overcome the risk of host immune response and the lack of helper-free production methods before they can be used extensively in the CNS (Lentz et al., 2012). On the other hand, the early generation of adenoviral vectors (Germano et al., 2003) as well as oncolytic adenovirus (Vera et al., 2016) may be used to treat brain tumors.

1.2.3 Adeno-Associated Virus

AAV belongs to the Parvoviridae family in the Dependovirus genus. It is nonpathogenic in humans. AAV virions are small nonenveloped particles (20–25 nm) that have an icosahedral protein capsid encompassing an about 4.7 kb single-stranded DNA genome (Atchison, Casto, & Hammon, 1965; Hoggan, Blacklow, & Rowe, 1966). The AAV genome contains two genes, rep and cap, flanked by T-shaped inverted terminal repeats (ITRs) (Lusby, Fife, & Berns, 1980). The rep gene encodes four regulatory Rep proteins (Rep78, Re68, Rep52, and Rep40) that are required for replication and packaging. The cap gene produces three structural proteins (VP1, VP2, and VP3) that form virion capsid (Goncalves, 2005) and the assembly activating protein, which is required for AAV capsid assembly (Sonntag et al., 2011). The ITRs are the only cis-acting elements that are necessary for genome replication, integration, and packaging into the capsid; these are the only genetic elements from the virus that are retained in recombinant AAV (rAAV) vectors. Replication of AAV is dependent on coinfection of a helper virus such as adenovirus and herpes virus. In the absence of a helper virus, wild-type AAV can establish a latent infection with a low frequency of integration at a specific region on chromosome 19. In rAAV vectors, the site specificity of integration is lost due to the absence of Rep (McCarty et al., 2004).

AAV vectors provide high transduction efficiency, robust long-lasting expression of transgenes, and low toxicity, making them very useful tools in animal models of human disease and in human gene therapy applications. The AAV vectors derived from AAV serotype 2 (AAV2) were the first vectors used for gene delivery in the CNS (Kaplitt et al., 1994; Klein et al., 1998; McCown, Xiao, Li, Breese, & Samulski, 1996). AAV2 transduces only a limited region after injection into the brain, which restricts its applications (Lo et al., 1999; Taymans et al., 2007). Many new serotypes of AAV have been isolated (Gao, Vandenberghe, & Wilson, 2005; Wu, Asokan, & Samulski, 2006). AAV serotypes are determined by AAV capsid. A new serotype is a newly isolated virus that does not efficiently cross-react with neutralizing sera specific for all other existing and characterized serotypes. The different AAV serotypes recognize different cell surface receptors and utilize a variety of approaches for cell entry and intracellular trafficking. Therefore, these serotypes give rise to distinct nervous cell tropism as well as distribution and strength of transgene expression. Non-AAV2 vectors are commonly produced using the ITRs sequences as well rep gene from AAV2 with only the cap gene from the desired serotype. This method is referred to as cross-packaging or pseudotyping. For example, a transgene containing AAV2 ITRs can be packaged with AAV8 capsid. The resulting AAV vector can be designated as AAV2/8 or simply called AAV8. In mouse brain, Taymans and colleagues reported that AAV serotypes 1, 2, 5, 7, and 8 direct gene expression primarily in neurons; these serotypes transduced comparable brain volumes in all targeted regions except for AAV2, which transduced smaller brain volumes (Taymans et al., 2007). Broekman et al. showed that AAV8 is more efficient than AAV1 and 2 for gene delivery to all areas in mouse brain (Broekman, Comer, Hyman, & Sena-Esteves, 2006). Cearley et al. showed that AAV7, 8, 9, and rh.10 vectors expressing cDNA for β-glucuronidase transduce neurons, but not astrocytes or oligodendrocytes, in the cortex, striatum, hippocampus, and thalamus (Cearley & Wolfe, 2006). While some serotypes transduce only within a normal range of diffusion, others also transduce cells at a substantial distance from the injection site via either anterograde or retrograde direction along axons. When injected into ventral tegmental area (VTA), AAV9 vector was the most widely disseminated, but AAVrh.10 and AAV1 were also transported after VTA injection (Cearley & Wolfe, 2007). Both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain (Castle, Gershenson, Giles, Holzbaur, & Wolfe, 2014). AAV1 has been shown to transduce spinal neurons through anterograde axonal transport after intracortical injection (Hutson, Kathe, & Moon, 2016).

There is also a great deal of interest in vectors that are able to cross the blood–brain barrier (BBB), so that a gene therapy can be achieved without having to inject vectors directly into the CNS. Foust and colleagues first demonstrated that intravenous injection of AAV9 can bypass the BBB and transduce cells in the CNS in both neonatal and adult animals (Foust et al., 2009). Duque et al. (2009) have shown motor neuron transduction in adult animals following intravenous delivery of self-complementary AAV9 vectors. Several other serotypes of AAV were found to be able to cross the BBB (H. Zhang et al., 2011). Among them, AAVrh.10 is at least as efficient as AAV9 in many of the regions examined. Besides naturally occurring AAV serotypes, numerous new AAV vectors have been developed through capsid engineering to increase targeting specificity and transduction efficiency. Phosphorylation of tyrosine residues on the AAV2 capsids has been found to negatively impact the intracellular trafficking of virus to the nucleus and transgene expression. Mutation of surface-exposed tyrosine residues leads to increased transduction efficiency both in vitro and in vivo (Zhong et al., 2008). This approach applies to other AAV serotypes as well. The AAV serotypes 8 and 9 mutant vectors display a strong and widespread transgene expression in many retinal cells after subretinal or intravitreal delivery compared with their wild-type counterparts (Petrs-Silva et al., 2009). Libraries of hybrid capsids from different wild-type viruses can be created. Gray et al. (2010) have used DNA shuffling and directed evolution to create novel AAV vectors to specifically target a therapeutic gene to sites of seizure damage in an animal model. By means of an in vivo–directed evolution approach, Dalkara et al. (2013) have created three types of capsid libraries (mutation, shuffling, and random insertion) and identified an AAV variant (7m8) that mediates highly efficient delivery to all retinal layers in mice and nonhuman primates. Deverman et al. (2016) have delivered AAV libraries to animals with Cre expression in a defined cell population and selectively recover capsids that transduce the Cre-expressing target cell. They found that one variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard AAV9 and transduces the majority of astrocytes and neurons across multiple CNS regions.

AAV vectors possess the advantages of efficient gene transfer, long-term transgene expression, minimal toxicity, and low immunogenicity. These properties make AAV vectors the currently preferred gene delivery vehicle for CNS applications. One of the major limitations for the use of AAV as a gene delivery vehicle is the small packaging capacity, which makes inclusion of large transgenes or cell-specific transcriptional regulatory elements difficult or impossible. The ability of AAV vectors to concatemerize by trans-splicing or homologous recombination, or a hybrid of the two mechanisms has been explored. Dural and tri-AAV vectors have been developed for expressing larger genes of interest. Despite a low efficiency of reconstitution, a full-length human dystrophin expression has been detected in tri-AAV vectors co-transduced tibialis anterior muscles of mdx4cv mice (Lostal, Kodippili, Yue, & Duan, 2014).

1.2.4 Herpes Simplex Virus

HSV is a member of Herpesviridae family with large double-stranded DNA genomes (Roizman, Knipe, & Whitley, 2007). HSV is neurotropic in vivo. It shows highly efficient retrograde and anterograde transport within the nervous system, making it an excellent candidate vector for gene delivery to the peripheral and CNS. Upon cellular entry, HSV can establish either a latent or lytic state. In the latent state, HSV exists as an extrachromosomal episome within the host nucleus. In the lytic state, HSV replicates, generating more viral particles and ultimately killing the infected host cells. Recombinant HSV vectors can be developed that are replication-defective, nontoxic, and capable of long-term transgene expression (Burton, Bai, Goins, & Glorioso, 2002). With multiple deletions in the virus genome, recombinant HSV can harbor large transgenes (30–50 kb). Recombinant HSV vectors still retain large proportions of the virus genome. Expression of viral genes can induce cytotoxicity and immune responses. HSV amplicon vector constructs have been developed, which contain an origin of viral DNA replication and a virion-packaging signal. Besides these cis-acting elements, no other viral genes are retained in the vectors. The large packaging capacity of HSV amplicons (about 150 kb) may be very useful for delivering complex genes and regulatory sequences (Sena-Esteves, Saeki, Fraefel, & Breakefield, 2000; Wade-Martins, Smith, Tyminski, Chiocca, & Saeki, 2001). The lack of viral genes also reduces toxicity of the vectors. Originally, HSV amplicon vectors were packaged into virions using a helper virus–based technology (Spaete & Frenkel, 1982). Helper-contaminated vectors can induce significant inflammatory responses as well as cytotoxicity. To overcome this problem, helper-free packaging systems have been developed. The HSV genes can been supplied in trans through artificial chromosomes (Saeki, Breakefield, & Chiocca, 2003) or by employing the Cre/loxP-based site-specific recombination to remove helper virus (Logvinoff & Epstein, 2001).

Several properties of HSV, including latency, neurospecificity, neuroinvasiveness, and very large packaging capacity, make HSV-derived vectors useful in the treatment of a wide range of disorders such as Alzheimer’s disease (Hong, Goins, Goss, Burton, & Glorioso, 2006), lysosomal storage disorders (Martino et al., 2005), and chronic pain (Goss, Krisky, Wechuck, & Wolfe, 2014). Replication-conditional vectors have been applied in the clinic to treat brain tumors (Markert et al., 2000, 2009). The shortcomings of HSV vectors include the duration of transgene expression, the potential for harmful side effects associated with co-production of helper virus, and the lack of a scalable system for vector production (Lentz et al., 2012).

1.3 Nonviral Vectors

Naked plasmid DNA may be the simplest, cheapest, and least toxic material to carry out gene transfer. Gene expression from plasmids is transient, which may be of benefit for some applications when only short-term transgene expression is desirable. Compared to viral vectors, plasmid DNA reduces the safety issues such as high frequency of insertional mutagenesis and an immune response to viral gene products. However, the transfection efficiency of naked DNA into cells, especially neurons, is extremely low and reagents used in transfection may be toxic. The nucleic acids are susceptible to nuclease-mediated degradation. High molecular weight and negative charges make the uptake of DNA into cells and subsequently trafficking to the nucleus extremely difficult (Takakura, Nishikawa, Yamashita, & Hashida, 2001). Nonviral gene delivery is usually coupled DNA with reagents that can help gene delivery and expression.

1.3.1 Nanoparticles

One way to reduce degradation of plasmid DNA and increase delivery to target cells is to use lipids or synthetic polymers to encapsulate plasmid and protect it from degradation leading to a more stable and efficient gene delivery. Liposomes containing polyethylene glycol (PEGylated) show decreased uptake by mononuclear phagocytic system and increased circulation half-life in vivo (Allen, Hansen, Martin, Redemann & Yau-Young, 1991). Both lipid-based and polymer-based nanoparticles have been widely used for gene delivery (Keles, Song, Du, Dong, & Lin, 2016). Hybrid nanoparticles can be generated with incorporating various ligands to increase their targeting efficiency and specificity. This is especially important for gene delivery into the CNS as the presence of BBB precludes the entry of most therapeutic agents from blood to the brain, which makes vector delivery by systemic administration a big challenge (Pardridge, 2005). A short peptide derived from rabies virus glycoprotein (RVG) enables the transvascular delivery of small interfering RNA (siRNA) to neuronal cells in the brain, resulting in specific gene silencing (Kumar et al., 2007). Liu et al. (2016) generated nanoparticles containing an amino-acid inhibitor of tau fibrils (D-peptide) and a short hairpin RNA (shRNA) coding plasmid targeting the β-site APP cleaving enzyme 1 antisense (BACE1-AS) transcript. The D-peptide and brain-targeting RVG29 peptide were covalently linked to dendrigraft poly-L-lysines and then encapsulated into the nanoparticles along with the therapeutic plasmid. Upon delivery of the nanoparticles into mouse model of Alzheimer’s disease (AD), down-regulation of BACE1 enzyme and reduction of neurofibrillary tangles were achieved. Similarly, Conceicao et al. reported that stable nucleic acid lipid particles incorporating a short peptide derived from rabies virus glycoprotein (RVG-9r) and encapsulating siRNAs targeting mutant ataxin-3 gene product efficiently silenced mutant ataxin-3 and reduced neuropathology and motor behavior deficits in two mouse models of spinocerebellar ataxia type 3 (Conceicao et al., 2016). Monoclonal antibodies specifically binding to receptors located on both the BBB and on brain cellular membranes (e.g., transferrin receptor) can also be incorporated into liposomes for nonviral gene delivery into the CNS (Boado & Pardridge, 2011). This Trojan horse liposome technology has been applied to animal models of lysosomal storage disorders (Zhang et al., 2008) and Parkinson’s disease (PD) (Zhang et al., 2004).

The microbubbles (MB) developed for ultrasound imaging in conjunction with focused ultrasound (FUS) bursts were found to produce reversible BBB disruption without brain tissue damage (Hynynen, 2008). Cationic MBs containing plasmid DNA or other forms of gene therapy agents can be generated and delivered to the brain through intravenous injection followed by FUS exposure. The FUS-triggered GDNF (glial cell-derived neurotrophic factor) plasmid-loaded cationic MB platform can achieve nonviral targeted gene delivery via a noninvasive administration route and has a neuroprotective effect in an animal model of PD (Fan et al., 2016). Mead et al. reported that co-injection of highly compact DNA-bearing brain penetrating nanoparticles (DNA-BPN) and MB into rat tail vein followed by applying magnetic resonance image–guided FUS resulted in dose-dependent transgene expression only in the FUS-treated region that was evident as early as 24 hours post administration and lasted for at least 28 days. In the FUS-treated region, about 42% of all cells, including neurons and astrocytes, were transfected (Mead et al., 2016). There are some limitations for these nonviral vectors. Gene transfer by nonviral vectors is generally not as efficient as viral vectors. Expression of genes delivered by plasmid-based vectors is transient, and repeated direct injection of vectors into the brain parenchyma is not practical. This precludes their application to many diseases in which sustained expression of the transgene is