Edited by: Ioanna Andreadou, National and Kapodistrian University of Athens, Greece
Reviewed by: Michelino Di Rosa, University of Catania, Italy; Kyle David Fink, University of California, Davis, United States
*Correspondence: Selene Ingusci,
This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology
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Neurological disorders affecting the central nervous system (CNS) are still incompletely understood. Many of these disorders lack a cure and are seeking more specific and effective treatments. In fact, in spite of advancements in knowledge of the CNS function, the treatment of neurological disorders with modern medical and surgical approaches remains difficult for many reasons, such as the complexity of the CNS, the limited regenerative capacity of the tissue, and the difficulty in conveying conventional drugs to the organ due to the blood–brain barrier. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. Gene therapy can result in a stable or inducible expression of transgene(s), and can allow a nearly specific expression in target cells. In this review, we will discuss the most commonly used tools for the delivery of genetic material in the CNS, including viral and non-viral vectors; their main applications; their advantages and disadvantages. We will discuss mechanisms of genetic regulation through cell-specific and inducible promoters, which allow to express gene products only in specific cells and to control their transcriptional activation. In addition, we will describe the applications to CNS diseases of post-transcriptional regulation systems (RNA interference); of systems allowing spatial or temporal control of expression [optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)]; and of gene editing technologies (CRISPR/Cas9, Zinc finger proteins). Particular attention will be reserved to viral vectors derived from herpes simplex type 1, a potential tool for the delivery and expression of multiple transgene cassettes simultaneously.
Even if scientific research has made great progress over the last decade in identifying pathogenic mechanisms and treatment strategies, neurological disorders affecting the central nervous system (CNS) are still incompletely understood. The majority of these disorders lack a cure or, at least, reasonably effective treatments. Reasons are certainly multifold and include the complexity of the CNS, the limited regenerative capacity of the tissue, and the difficulty in conveying conventional drugs to the organ across the blood–brain barrier (BBB). Neurons, the principal cells of the nervous tissue, are not only morphologically and physiologically heterogeneous, but also strictly organized to form complex circuits. Neural stem cells ensure only a limited replacement of only specific neuronal types. The BBB expresses a selective permeability for molecules that possess a limited range of molecular weight and lipophilicity, preventing the entry of large-molecule drugs and of the majority of small-molecule drugs.
In this context, gene therapy is emerging as an attractive therapeutic option, because it can result in a stable or inducible expression of therapeutic gene(s), and can allow a nearly specific expression in target cells. Although much remains to be done before it becomes routine practice, the potential of gene therapy for the treatment of CNS diseases is amply demonstrated by numerous preclinical and clinical studies (
A large part of the work needed to finally reach the stage of clinical application consists in the refinement of the tools needed for a safe, targeted, and regulated gene delivery. In this review, we will discuss the most commonly used gene therapy tools for delivery in the CNS and the strategies that can be employed for regulating therapeutic gene expression.
The idea behind gene therapy derives from an assumption of great simplicity: by introducing into the cells the “correct” copy of a defective gene whose malfunction causes a disease, its product, a functional protein, will be able to revert the pathological phenotype. This assumption may be correct for monogenic diseases caused by alterations in the coding sequence or in regulatory regions of a single gene, and if these alterations lead to loss of function without production of a pathogenic protein. However, many diseases have a pattern of multiple altered genes. Moreover, the regulation of gene expression is often complex and difficult to reconstruct. According to a new, broader definition, all drugs that contain an active substance that includes or consists of a recombinant nucleic acid (DNA or RNA), administered to a human being for the purpose to adjust, repair, replace, add, or remove a gene sequence, can be defined gene therapy (
The introduction of a functional gene, called transgene, within the cell nucleus is a complex operation that starts with the choice of a delivery system (gene therapy vector). A good vector should fulfill many requirements (
Manipulation: the vector should be easily manipulated for recombination and propagation in suitable hosts.
High cloning capacity: the vector should allow the introduction of one or more genes and regulatory sequences that guarantee the desired spatial and temporal restriction of transgene expression.
Minimal invasiveness: the vector should not cause uncontrolled or undesired alterations of the host genome. The integration of a vector into the cellular genome can induce insertional mutagenesis.
Selectivity for the cellular target: the transgene should be expressed exclusively in the target cells.
Absence of immunogenicity: the vector should not contain genes that induce immune responses or other factors that may be harmful to the body.
Stability over time: the vector should be transferred unaltered in the cell progeny and/or must allow a correct and prolonged expression of the transgene(s).
Available gene therapy vectors belong to two broad categories: viral and nonviral vectors.
Nonviral vectors offer some advantages, like reduced pathogenicity, low cost, and simple production techniques (
In addition to direct injection and microinjection of DNA into the nucleus, physical methods and chemical carriers have been developed to improve delivery of naked DNA to cells and tissues (
Independent of their origin, order, and family, viruses have evolved very fine strategies to reach and penetrate specific cellular targets. Their use in gene therapy lies in their innate ability to deliver and express genetic information into host cells. Replication-defective viral vectors (
Main features of the most commonly employed viral vectors for CNS gene therapy.
Viral vector | Payload | Tropism |
---|---|---|
|
up to 9 kb | Proliferating and quiescent cells |
|
7–10 kb |
Dividing and non-dividing cells |
|
∼4.8 kb | Different serotypes with different tropism; typical is tropism for hepatocytes, myocytes, and neuronal cells |
|
∼40 kb |
Actively dividing tumor cells like glioblastoma, hepatocellular carcinoma or melanoma cells |
The
While the lentiviral integrative nature ensures stable and persistent expression of the transgene, it also entails the risk of insertional mutagenesis. However, gene editing allowed to develop safe lentiviral vectors with specific integration sites (
Since lentiviral vectors transduce neurons effectively, they have been tested for the treatment of Alzheimer’s disease (AD) and Parkinson’s disease (PD) (
Adenoviruses are linear double-stranded DNA viruses with a genome size of 35–40 kb encoding approximately 30–40 genes. There are 100 serotypes of adenovirus, 57 of which have the potential to infect humans. These are divided into seven subgroups, A to G, that differ in cellular tropism (
Adenoviral vectors transduce efficiently dividing and nondividing cells, with no risk of integration in the host cell genome (
Adenoviral vectors have been widely studied for the treatment of tumors (
Adeno-associated viruses (AAVs) are small, non-enveloped, single-stranded DNA viruses belonging to the
More than 12 different AAV serotypes have been isolated (
Owing to the small sized genome, AAV vectors are capable of accommodating less than 5 kb of exogenous DNA (
The therapeutic potential of AAV-based gene therapy has been tested in many different neurological disorders (
The first Herpesvirus vector has been derived from Herpes Simplex Virus type 1 (HSV-1) (
The large genome (152 kb) of HSV-1 encodes about 80 genes, half of which can be removed to make room for up to 50 kb of foreign DNA in the case of replication-defective vectors and up to 150 kb for amplicon vectors (
Disorders of the CNS are often not a result of single gene mutation or of a single molecular mechanism but have instead a multifactorial origin. As a result, the therapeutic gene(s) and/or the regulation sequence to be delivered very often exceed the payload capacity of viral vectors (
The HSV productive cycle is characterized by a temporally regulated cascade of gene expression, during which three distinct classes of transcripts are expressed in a sequential manner. The immediate early genes (IE or α) are first expressed, followed by the early (E or β) and late (L or γ) genes. IE genes are required not only for establishment of a lytic reproductive cycle, but also to overcome innate immune responses, to block cell division, and to prevent host cell apoptosis and epigenetic repression of viral genes. The engineering of mutant HSV-1 vectors devoid of α genes shuts off viral replication and remarkably reduce cytotoxicity (
Schematic drawing of the genomes of new HSV-1 vectors. Compared to the wild-type genome
Amplicon vectors (
Schematic representation of an amplicon vector. The amplicon plasmid contains the sequences for viral DNA replication (ori, blue) and the packaging signal (pac, red), flanking the transgene expression cassette. In the presence of an HSV-1 helper virus in permissive cells, the amplicon DNA plasmid is replicated as head-to-tail concatemers, cleaved into 150 kb linear DNA and packaged in HSV capsids.
The question that comes together with the development of suitable vectors for gene transfer in the CNS is which genes to transfer. The first, most obvious option is genes encoding a therapeutic protein. This may be the correct copy of a defective gene whose malfunction causes the disease, but also a gene encoding a therapeutic protein that could not be peripherally administered, not being able to cross the BBB. In this last instance, the protein could be diffusible (i.e., produced and secreted by the infected cell) to produce a by-stander effect in adjacent cells (
Apart from genes encoding therapeutic proteins, however, there are other cargo options for CNS gene therapy vectors, like gene editing, chemogenetic, and optogenetic tools.
A step forward was made in gene therapy with the development of gene editing tools that can correct genetic defects directly in the host DNA. These tools generate a double strand break (DSB) at a precisely desired location, and the break allows to take advantage of the fine strategies that cells have evolved to detect and repair DNA damage. The DSB can lead to gene disruption by non-homologous end joint (
The complexity of the mammalian brain has no comparison: dozens of billions of interconnected neurons, with complex morphology and circuit interaction, capable of exchanging electrical signals with a precise temporal scan in the order of milliseconds. A great challenge is to develop the ability to control only one type of cell in the brain without affecting others. Electrical, physical, pharmacological, and genetic methods are traditionally used to manipulate cells and synapses (
Chemogenetics is the processes in which proteins are engineered to interact specifically with a small molecule (
Together with optogenetics, the DREADDs technology is currently the most used tool for
DREADDs are useful tools for basic scientific research but may also refine gene therapy approaches for neurodegenerative disorders in which changes in neuronal activity play an important role. Neuronal hyperactivity and hyperexcitability of the cerebral cortex and hippocampus are common features of epilepsy and AD (
The term optogenetics indicates a methodology that allows to control the activity of specific neurons within intact neuronal circuits (
Channelrhodopsin (ChR): a blue light activated cation-channel from
Halorhodopsin (NpHR): a yellow light activated chloride-pump from
Through viral vectors, the gene coding for an opsin can be integrated into target neurons, leading to expression of the opsin protein on the membrane. A nearby source of light, set on the right wavelength and frequency, can then interact with it, activating or inhibiting neuronal activity. The introduction of mutations to existing opsin variants allowed to overcome certain problems associated with light delivery. For example, the ChR chETA mutant displays faster channel closing and increased temporal control (
The use of optogenetics as therapeutic tools for neurological disorders has been investigated in PD, AD, and epilepsy (
As previously mentioned, precise regulation of gene expression is essential for any gene therapy approach. A good gene regulation system should be adjustable over a broad dose range; should exert no off-target effect; does not influence endogenous gene expression; should be region or cell specific; and should allow to quickly turn off and on transgene expression (
Regulation or RNA transcription depends on the euchromatin and heterochromatin state and on the interaction of transcription factors with regulatory DNA elements, including promoters (
Regulation of gene expression at transcriptional level.
Promoter | Specificity | Size (bp) | Details | References |
---|---|---|---|---|
CAG | Ubiquitous | 1,718 | Hybrid construct consisting of the cytomegalovirus (CMV) enhancer fused with the chicken beta-actin promoter | ( |
EF1α | Ubiquitous | 1,179 | Human elongation factor 1 alpha promoter | ( |
UBC | Ubiquitous | 1,177 | Human ubiquitin C promoter | ( |
SV40 | Ubiquitous | 627 | Simian virus 40 promoter | ( |
CMV | Ubiquitous | 589 | Human cytomegalovirus immediate early enhancer and promoter | ( |
PGK | Ubiquitous | 511 | Mouse phosphoglycerate kinase 1 promoter | ( |
Syn1 | Neuron | 495 | Human synapsin 1 promoter | ( |
NSE | Neuron | 1,800 | Neuron-specific enolase promoter | ( |
GFAP | Astrocytes | 681–2,200 | ( |
|
MAG | Oligodendrocytes | 1,500–2,200 | Human myelin associated glycoprotein | ( |
MBP | Oligodendrocytes | 1,900 | Myelin basic promoter | ( |
F4/80 | Microglia | 667 | ( |
|
CD68 | Microglia | 460 | ( |
|
PAG | Glutamatergic neurons | 2,400 | Phosphate-activated glutaminase promoter | ( |
vGLUT | Glutamatergic neurons | 7,000 | Vesicular glutamate transporter promoter | ( |
GAD | GABAergic neurons | 10,000 | Glutamic acid decarboxylase promoter | ( |
Tetracycline ON/OFF system | Inducible promoter | Advantages: rapid |
( |
|
Rapamycin regulation system | Inducible promoter | Advantages: low basal expression, trigger by low doses, crosses BBB. Limitations: immunosuppressive properties of rapamycin. | ( |
Promoters are the main elements that determine the strength and cellular specificity of gene expression. Ubiquitous and constitutive promoters are strongly active in a wide range of cells and tissues. Therefore, ubiquitous expression promoters are used in gene therapy when targeting a specific cell type is not required, i.e., transgene expression is sought in the broadest possible spectrum of cells. Promoters frequently employed to drive exogenous DNA expression in a non-cell specific manner include cytomegalovirus (CMV) immediate-early; enhancer/chicken-β actin (CAG); human ubiquitin C (UBC); simian virus 40 early (SV40); human elongation factor 1α (EF1α); and mouse phosphoglycerate kinase 1 (PGK). Previous works have described the relative strengths of commonly used transcriptional regulatory elements both
The use of cell-type specific promoters may be useful for confining the transgene expression to a specific cell type. Limitations for their use in gene therapy include their low level of expression and their large genomic size. In principle, however, specifically labeling a population of neurons or glial cells might allow to achieve the therapeutic goal without incurring in off-target effects. The synapsin-1 (Syn1) and the neuron-specific enolase (NSE) promoter are used for their ability to selectively drive transgene expression in neurons (
The balance between excitatory and inhibitory signals, basically the equilibrium between glutamatergic and GABAergic neurotransmission, can be often disrupted in diseases like epilepsy. Therefore, targeting specifically GABAergic or glutamatergic neurons may be needed for many applications. The phosphate-activated glutaminase (PAG) or the vesicular glutamate transporter (vGLUT) promoter ensures ∼90% glutamatergic neuron-specific expression, whereas the glutamic acid decarboxylase (GAD) promoter ensures ∼90% GABAergic neuron-specific expression (
Promoters are not the only elements necessary for transcriptional regulation. Combining regulation elements of different kinds such as promoters, enhancers, introns, and polyadenylation signals by creating hybrid sequences allows modulation of the expression levels. The levels of transgene expression may be strongly influenced by a rapid epigenetic silencing of the exogenous promoters. To protect the promoter and the whole expression cassette from heterochromatization, insulator elements have been tested for their ability to maintain transcriptionally competent whole portions of DNA, regardless of the tissue type and the integration site (
For many applications, it is desirable to modulate the expression of the transgene by switching it on or off. Unregulated long-term overexpression of certain transgenes can cause side effects in the CNS, such as aberrant reorganization of the tissue and activation of compensative pathways and/or inactivation/saturation of activated pathways. A finer regulation can be achieved using inducible promoters. These systems are obtained by incorporating in the vector (or in a separate vector) a cassette driving the constitutive expression of a transcription factor (transactivator) able to activate or block the expression of the transgene depending on the availability of a soluble molecule that can be administered systemically.
A commonly used regulation system is based on the mechanism of tetracycline resistance in prokaryotes. Two variants are available (
Tetracycline regulation system. In this example, the constitutively active human cytomegalovirus promoter (pCMV) drives the expression of the tetracycline transactivator (tTA) or of the tetracycline reverse transactivator (rtTA), consisting respectively of the tet-repressor (tetR) or reverse tet-repressor (rtetR) fused to the VP16 transactivation domain. Tet-off: tTA binds the tet operator (tetO) to drive transgene expression in the absence, but not in the presence of doxycycline (dox). Tet-on: rtTA binds to tetO and drives transgene expression in the presence, but not in the absence of dox.
Tetracycline-based regulatory systems hold a great potential for gene therapy applications. They ensure rapid
For CNS applications, it is necessary to obtain adequate concentrations of doxycycline in the brain, which is difficult due to the limited ability of this drug to cross the BBB (
The rapamycin regulation system is based on the interaction between two inactive transcription factors, a DNA binding domain and a DNA transcriptional activation domain. Each transcription factor is fused to heterologous binding domains for rapamycin. The DNA binding domain is fused to three copies of the FK-binding protein (FKBP), while the DNA activation domain is fused to a lipid kinase, FKBP12 rapamycin-associated protein (FRAP) (
Rapamycin regulation system. In this example, the constitutively active human cytomegalovirus promoter (pCMV) drives the expression of two transcription factors, one consisting of the FK-binding protein (FKBP) fused to a DNA binding domain (in green), the other consisting of an FKBP12 rapamycin-associated protein (FRAP) fused to a DNA activation domain (in red). Rapamycin enables dimerization of the transcription factors and the resulting heterodimer is able to drive expression of the transgene of interest.
This system holds many of the features required for clinical use. First, rapamycin is a clinically approved drug, used as an antifungal and antitumor molecule that can be administered orally and can cross the BBB (
A limited number of studies tested this system in the CNS by delivering it through lentiviral (
The term “post-transcriptional gene regulation” refers to approaches designed to enhance degradation or block translation of a target mRNA. The prototypical example is RNA interference (RNAi,
RNA interference-gene silencing pathways mediated by siRNA or miRNA. Even though siRNAs precursors are commonly delivered into the target cells as exogenous double-stranded RNAs
Neurodegenerative diseases resulting from a single gene mutation that causes gain-of-function or accumulation of a mutant protein are potential candidates for RNAi. Unfortunately, RNA molecules do not cross the BBB, thus requiring the use of viral or nonviral vectors for CNS delivery. This may result in widespread changes in expression levels in unrelated genes due to nonspecific degradation of nontarget mRNAs (
For example, RNAi has been investigated in HD, an autosomal-dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene (
New promising tools acting at a post-transcriptional level are short antisense oligonucleotides (ASO,
Gene silencing by delivery of antisense oligonucleotides (ASO). Cell-delivered ASOs can act as translation repressors with different mechanisms. If ASOs reach the nucleus
The rapid progress of viral and nonviral vector systems has increased the probability of success of CNS gene therapy as an alternative to existing pharmacological treatments. However, all the delivery systems developed thus far have advantages and disadvantages and, therefore, the search for an ideal one continues. A lesson learned from the research performed to date is that delivery tools do not necessarily adapt to all applications, but should be chosen according to the specific situation and need. Understanding the rules of transcriptional and post-transcriptional gene regulation will allow to improve our techniques. In addition, optogenetic and chemogenetic approaches can provide a precise temporal and spatial regulation of gene expression, and the recent introduction of genome-editing technologies allows the direct manipulation of the genome. Therefore, even if more work will be needed to overcome the remaining hurdles, gene therapy now holds a strong promise to become a safe and effective option for CNS diseases in the not too distant future.
SI, GV, and MSi wrote the largest part of the article. MSo wrote the chapter on amplicons, and SZ wrote the chapter on viral vectors.
This work was supported by grants from the European Community (FP7-PEOPLE-2011-IAPP project 285827 [EPIXCHANGE] and FP7-HEALTH project 602102 [EPITARGET]).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.