Gene Therapy Approaches for Neurological Disorders (CNS): progress and prospects
Abstract
Gene therapy for neurological disorders is currently an experimental
concept. The goals for clinical utilization are the relief of symptoms, slowing
of disease progression, and correction of genetic abnormalities. Experimental
studies are realizing these goals in the development of gene therapies in
animal models. Discoveries of the molecular basis of neurological disease and
advances in gene transfer systems have allowed focal and global delivery of
therapeutic genes for a wide variety of CNS
disorders. Limitations are still apparent, such as stability and regulation of transgenic
expression, and the safety of both vector and expressed transgene. In addition, the
brain adds several challenges not seen in peripheral gene therapy paradigms, such as post-mitotic cells, heterogeneity
of cell types and circuits, and limited access. Moreover, it is likely that
several modes of gene delivery will be necessary for successful gene therapies
of the CNS. Collaborative efforts
between clinicians and basic researchers will likely yield effective gene therapy in the CNS.
Introduction
Diseases of the central nervous system (CNS) have traditionally been the most
difficult to treat by traditional pharmacological methods, due mostly to the
blood-brain barrier and the difficulties associated with repeated drug
administration targeting the CNS.
Viral vector gene transfer represents a way to permanently provide a therapeutic protein within the nervous system after a single administration,
whether this is a gene replacement strategy for an inherited disorder or a
disease-modifying protein for a disease such as Parkinson’s. Gene therapy approaches for CNS disorders has evolved considerably
over the last two decades. Although a breakthrough treatment has remained elusive,
current strategies are now considerably safer and potentially much more
effective focusing on clinical trials utilizing adeno-associated virus and
lentiviral vectors.
Exogenous and endogenously
regulated gene therapies are each associated with a set of advantages and
disadvantages when considering their use in patients. Firstly, exogenously
regulated systems usually require long-term administration of a drug. This
necessitates extensive characterization of the safety and tolerability of the
drug, which, with the exception of tetracycline, has not occurred thus far.
Additionally, it is not possible to tailor gene expression precisely to
pathological signals using exogenous systems unless they are associated with a
noticeable phenotypic change in a timely manner. In most cases, there is a
latent period between the presence of a pathological signal and the appearance
of clinical symptoms. Therefore, endogenous systems have the advantage of
responding in a timelier manner and with an intensity that is tailored to the
individual patient.
Additionally, gene
expression systems are typically administered to whole organs or entire regions
of organs, and while it may be advantageous to express the transgene in this manner;
this is quite often not the case. By linking transgene expression to a
pathological signal, the recombinant protein will only be produced in cells that
require it, and toxic overexpression in healthy cells can be avoided.
Conversely, a major limitation of endogenous systems is that once administered
it will be virtually impossible to stop transgene expression in the event of an
adverse reaction, unless the population of cells of interest is removed, or
there is a built-in safety mechanism. Additionally, the pathological signals
used need to be as far upstream as possible and be specific to the disease
phenotype to enable transgene to be expressed in a timely and precise manner.
Genetic therapy covers a range of
methods for modifying the nervous system, including the delivery of genes,
sequence-targeted regulatory molecules, genetically modified cells and
oligonucleotides. In this review, we focus on ways to change the genetic status
of the nervous system, including routes of access and modes of delivery.
Examples are provided of strategies used for a number of diseases, including
recessive loss of function conditions [lysosomal storage diseases and spinal
muscular atrophy (SMA)], dominant toxic mutations [amyotrophic lateral
sclerosis (ALS)] and conditions of mixed etiology [Alzheimer’s disease (AD),
Parkinson’s disease (PD) and brain tumors] Neurodegenerative diseases caused by
triplet nucleotide repeats are discussed in an article on siRNAs in this issue.
Many of these strategies have proven very promising in preclinical studies in
mouse and larger animal models, and a substantial number are now being
evaluated in clinical trials.
Therapeutic strategies based on genetic
etiology. (A) Recessive disease. In the case of a recessively
inherited disease where both alleles (and one on the X chromosome in males) of
a gene are mutated, the goal is to replace the defective gene with a functional. Traditional pharmacological approaches
often run into considerable challenges when treating CNS disorders. It is difficult to get many compounds across the
blood-brain barrier (BBB). Even for compounds that cross the BBB, very large
doses must be administered into the blood to get enough of the drug into the
brain to be effective. This can often lead to side effects in peripheral organs
that must be considered. Methods to concentrate the drug within the nervous
system, such as intrathecal administration are possible but chronic
administration of the drug has a significant risk of complications.
The benefit of gene transfer is that the
therapeutic agent (protein, siRNA, etc.) can be produced within the CNS and provided on a permanent
steady-state basis after a single administration. AAV vector technology is
advancing rapidly, facilitating new approaches for clinically relevant gene
transfer. Changes to the capsid, genome design, and route of administration
have made global CNS gene transfer
possible in ways that are expected to translate to humans. This has led to a
renaissance of sorts in gene therapy
strategies to treat inherited neurological disorders.
Recent years have seen a plethora of
potential gene therapy strategies
for the central nervous system (CNS)
disorders. One of the major challenges gene
therapy applications face clinically is the ability to control the level of
expression or silencing of therapeutic genes in order to provide a balance
between therapeutic efficacy and nonspecific toxicity due to overexpression of
therapeutic protein or RNA interference-based sequences. Thus, the ability to
regulate gene expression is essential as it reduces the likelihood of
potentially initiating adverse events in patients. Although genes maybe
regulated at either the translational or posttranscriptional level, greatest
success in gene regulation has been at the transcriptional level and as such
gene regulation systems at a transcriptional level are the focus of this review.
There are two classes of gene regulation
systems—exogenously controlled gene regulation systems, which rely on an
external factor (usually the administration of a drug) to turn transgene the expression on or off, and endogenously controlled gene expression systems that
rely on physiological stimuli to control transgene expression. This review
covers the characteristics of an ideal regulatory system and summaries the
mechanics of current gene regulation systems and their application to CNS disorders
Related work discussion
The concept of gene delivery using
virus-derived vectors was introduced in the mid-twentieth century. In this
chapter, we review the different viral-based vectors (Retro-, Lenti-, Adeno- and
Adeno associated viruses) developed over this time in clinical trials for
treating different CNS pathologies including brain tumors, Parkinson’s disease,
Alzheimer’s disease, and cancer pain. We describe the successes achieved and
the challenges still faced for each of these viral vectors as well as brain
disease conditions. This approach was widely perceived as ground-breaking for
treating a wide spectrum of genetic diseases (Friedmann et al. 1976). The development of efficient viral vector platforms rapidly propelled human gene therapy to the forefront as a
means to correct otherwise fatal disorders. Simple retroviral vectors
(γ-retroviruses) were among the first that were utilized in preclinical and
clinical studies due to low immunogenicity, long-term transgene expression, and
a relatively simple manufacturing protocol.
The first proof-of-principle study
using γ-retroviruses to correct a genetic disease in humans was a trial
attempting to correct a severe combined immunodeficiency disorder (SCID)
carried out in Kenneth Culver’s laboratory (Blaese et al.1995). In this
clinical trial, CD34-positive cells were isolated from two patients with
inherited adenosine deaminase deficiency, transduced ex vivo with a
γ-retrovirus, which carried the normal version of the gene, and readministered
to the patients. One patient exhibited a temporary response, although she
continued on enzyme replacement therapy (ERT). The response was far more
limited in the second patient. Similar clinical trials were later conducted by
Alain Fischer’s group in France (Cavazzana-Calvo et al. 2000). (Gaspar et al.
2004).
Tragically, in both clinical trials,
several children developed T-cell leukemia within 2–5 years after gene therapy, and one of these children
died. Analysis of the patients with leukemia revealed intentional mutagenesis
in the leukemic T-cell clone, which was correlated with the onset of leukemia.
Integration of the provirus resulted in up-regulation of adjacent proto-ontogenesis
due to the strong promoter elements in the γ-retrovirus long terminal repeats
(LTR). Until this incidence, the risk of inspectional mutagenesis of
retroviruses was estimated to be only 10−6–10−8 per integration event (Stocking
et al., 1993). Currently, the frequency for a transforming insertion in a
region of 10 kb around a proto-oncogene is calculated as 10−2–10−3 (Baum et al.
2003). Which highlights the limitations of the γ-retroviral vector approach.
HSV-1 recombinant virus and amplicon
vectors Herpes simplex virus type 1 (HSV) is a common pathogen in humans,
causing primarily cold sores, but occasionally encephalitis and other
life-threatening conditions, especially in immune-compromised individuals. It
is an enveloped virus bearing 152 kb of double-stranded DNA encoding over 80
genes, which has high infectivity for neurons and glia, as well as many other
cell types.
The virion enters the cell by fusion of
the envelope with the plasma membrane, and the capsid is transported along
microtubules to the nucleus. In neurons, HSV vectors are delivered by rapid
retrograde transport along neurites to the cell body, providing a means of
targeting gene transfer to cells that are difficult to reach directly. The
viral DNA is deposited in the nucleus, initially in a circularized episomal
form, and eventually replicates, enters latency or is degraded depending on its
composition. Two types of vectors are derived from HSV: recombinant virus
vectors (RV) and amplicon vectors (Glorioso et al. 2007).HSV-RV vectors contain
the full viral genome mutated in one or more virus genes to reduce toxicity and
provide space for transgenes (30–50 kb).
Replication-conditional RV vectors can
selectively replicate in and kill tumor cells in the brain. Replication-defective
RV vectors are designed to have minimal toxicity, and current versions delete
multiple immediate–early genes that encode transactivating factors, thereby
essentially eliminating expression of other viral genes, eg deletions of genes
encoding ICP4, ICP22, ICP27 and ICP47 (the latter being involved in antigen
presentation). Elimination of ICP0 further reduces toxicity in some cells but
also results in low levels of transgene expression.RV vectors can enter a
stable, benign, episomal latent state in neurons, but with consequent
down-regulation of most viral and cellular promoters. The long-term expression has
been achieved in neurons using the LAT promoter(s) which are active in viral
latency. The HSV amplicon vector consists of a plasmid bearing the HSV origin
of DNA replication, oris, and packaging signal, pac, which allows it to be
packaged as a concatenate in HSV virions in the presence of HSV helper
functions. These vectors can be packaged free of helper HSV virus by
cotransfection with the HSV genome deleted for space signals using a set of
cosmids or BAC plasmid.
The likelihood of
insertional mutagenesis might be lower when utilizing lentiviral vectors. For
example, in a model with tumor-susceptible mice, transplantation of
γ-retroviral vector-transduced hematopoietic cells resulted in an accelerated
tumorigenic process, whereas no additional adverse events were detected with
lentiviral vectors (Montini et al. 2006). Furthermore, it has been demonstrated
that a higher quantity of lentiviral vectors is necessary to cause an oncogenic
risk similar to that of γ-retroviral vectors (Montini et al. 2009). Thus, the
use of lentiviral vectors should provide significant advantages in reducing the
potential for adverse mutagenic events. Interestingly, this problem had already
been taken into consideration from a theoretical point of view before the
clinical trials mentioned above (Cline et al.1985) (Hacein et
al.2003)(Hacein-Bey-Abina et al. 2003).
Another major disadvantage of using γ-retroviral vectors is the fact that they only transduce
dividing cells. The infection of nondividing cells is possible, but the nuclear
membrane must be disassembled for the integration of the viral cDNA into the host cell genome (Lewis et al.1994). (Miller,et al.1990). Thus, in order to
target nondividing or terminally differentiated cells (e.g., post-mitotic
neurons) lentiviral vectors should be employed. Nuclear import of the
lentiviral genome is maintained by the host proteins (Lewis et al. 1994).
Efficient transduction of neuronal cells in vivo was shown in the very first
publication that utilized a lentiviral platform for the gene delivery (Naldini
et al. 1996). Lentiviral vectors also have been shown to transduce most cell
types within the CNS in vitro and in
vivo, including premitotic and postmitotic neurons, adult neuronal stem cells,
astrocytes, and oligodendrocytes (Blomer et al. 1997)( Consiglio et al. 2004).
The
first clinical trials that employed lentiviral vectors to treat inherited
disorders for adrenoleukodystrophy (ALD) (Phase I/II) (Cartier et al., 2009)
and β-thalassemia (currently Phase III) were conducted in Europe. These
clinical trials provided evidence of therapeutic efficacy in several patients
for at least 6 years. Another clinical trial utilized lentiviral vectors for the delivery
of multiple genes involved in dopamine biosynthesis. This vector (ProSavin) is
currently being tested in Phase I/II trials for PD (Grosset, 2010)( Stewart et
al. 2011). Finally, two more recent trials utilized lentiviral vectors for gene therapy of inherited diseases;
metachromatic leukodystrophy (MLD) (Biffi et al., 2013) (Phase I/II), and
Wiskott–Aldrich syndrome (WAS) (Aiuti et al. 2013) (Phase I/II) are discussed
in this chapter.
CONCLUSION
Promising results have been obtained in small clinical trials for
MLD and WAS using lentiviral vectors by ex vivo gene transfer approaches, but a
clear understanding of the extent of rescue and confirmation of these
preliminary results will require larger clinical trials. Early trials also
identified the risk of insertional mutagenesis, ultimately leading to
oncogenesis. Emerging technology for lentiviral vectors appears to overcome
many of the early safety concerns for retroviral vectors.
For AAV vectors, early clinical trials demonstrated successfully
gene transfer, but large impacts on the disease symptoms were lacking except
perhaps for the small AADC-deficiency trial. Clinical trials for PD have shown
encouraging results, but are plagued by a placebo effect that has made
assessing the potential of gene therapy
difficult. LSDs represent a promising family of diseases that could benefit
from gene therapy. The main obstacle
in the translation of LSD gene therapies has been the availability of a global
gene delivery system applicable to large animals; however, promising
technological developments utilizing IV or intra-CSF AAV vector delivery are
beginning to meet that need.
In summary, the clinical trials to
date have laid important groundwork in the advancement of CNS-directed gene therapy.
While an unequivocal clinical success for CNS
gene therapy has remained elusive,
upcoming clinical trials will be testing approaches that have much greater
potential to successfully translate encouraging results from animal models into
humans. In the upcoming years, clinical trials for SMA, GAN, MPS IIIB, and
other diseases will test the promise of global AAV-mediated CNS gene transfer. Concurrently, the
promising results from the MLD and WAS trials using lentiviral vectors are
being translated to other related diseases. Once a gene therapy breakthrough is realized for one disease, no matter
how rare, the vector platform and approach can serve as a template for the
treatment of a wide range of neurological disorders.