in Gene Therapy for the Treatment of Neurological Disorders
Motivation:
Gene
Therapy is a latest technology by which diseases are treated by introducing
genes. It is especially used to treat neurological disorders. Over the past 20
years more development has occurred in using gene therapy in clinical field to
cure neurological disorders. It has become a hopeful for treatment of a number
of CNS disorders which were previously considered as incurable. Clinical trials
have been performed for the past 20 years to cure diseases such as Lysosomal
Storage Diseases, Alzheimer’s disease, Parkinson’s disease, Spinal Muscular
Atrophy. Especially to cure Brain Tumors a lot of trials are being done and in
process. There are different approaches which are utilized in gene therapy. It
involves replacement of gene for a particular inherited disorder or
modification of the defective gene in some disorders. By using gene therapy
techniques nervous system is modified by different methods which include
delivery of specific genes and sequence targeted molecules. Gene Therapy
utilizes Recombinant Viral Vectors which serve as vehicles for genes transfer
and the most efficient means for gene expression in CNS. These viral vectors
include Adeno-Associated Viral Vectors (AAV), Retroviral Vectors, Adeno Viruses
and Herpes Simplex Viral Vector (HSV). These Vectors are modified in such a way
that they become nontoxic and carry desired genes which are used to cure a
disease. These vectors are inserted into the CNS either directly into brain
parenchyma tissue or via CSF or may be through intra vascular pathway.
Non-viral vectors are also being made now a day. These are DNA or RNA packed in
organically modified silica based nanoparticles. Two gene based drugs Gendicine
and Glybera are being made so far and are available in the market. More
development is being made in the field of gene therapy but there is still need
of a breakthrough so that it becomes common and used to cure all neurological
diseases.
Key
Words: Central Nervous System (CNS), Gene Therapy, Blood
Brain Barrier (BBB), Alzheimer’s disease (AD), Parkinson’s disease (PD),
Epilepsy, Amyotrophic Lateral Sclerosis (ALS), Adeno-associated Viruses (AAV),
Retroviruses, Adenoviruses, Herpes Viruses.
Results:
gene
therapy is ideal for curing CNS disorders than using traditional medicines but
still more research is needed in this field. A breakthrough is still needed for
the treatment become common hence, it is concluded that more work should be
done in the field of gene therapy.
Introduction
Diseases of Central Nervous System (CNS) are caused
by inherited genetic mutations which cause neurodegeneration or impaired
functions.
neurodegenerative diseases are caused by unfolding of proteins.
of CNS diseases are disease related events, injury or inflammation. Traditional
medical and surgical procedures don’t give effective treatment.
Simonato, may, 2013)
proteins from blood into CNS is limited by Blood Brain Barrier (BBB) &
skeletal structures enclosing it. BBB has tight junctions between brain
microvascular endothelial cells which limit the movement of substances through
it.
contributed in this regard to treat these neurological disorders.
Simonato, may, 2013)
for the treatment of human diseases was given during 1980s.
of Gene Therapy to treat human diseases was given by Friedmann & Roblin in
1972.
such as Alzheimer’s Disease(AD), Parkinson’s Disease(PD), Epilepsy, Amyotrophic
Lateral Sclerosis(ALS) have been studied to be cured using Gene Therapy.
been a major problem in neurologinal disorders. Attempts have also been made to
cure glioma using gene therapy techniques.
has been a great problem for people has been treated using multiple gene
therapy techniques.
autosomal recessive neurogenerative disorder of CNS also treated by gene
therapy.
In gene therapy genes are inserted into target cells
by using vectors.
vectors to deliver genes to cells of CNS. These viruses are Adeno-associated Viruses
(AAV), Retroviruses, Adenoviruses & Herpes Viruses. Viruses are simplest
organisms which have properties to be used as vectors for delivering of genes
to cells. The structure of a virion carries & protects the genetic
information (DNA & RNA) which provides the stability to this genetic
material. Viruses have determinants which specify cells in which viruses will
transfer specific gene. Viruses can express genes in the infected cells.
Viruses can replicate within the host cell so that the desired gene also
replicates in the host cell. Ideal vectors are those which do not carry genes
of disease but only have desired genes. AAV is a good viral vector as it is
easily prepared, do not cause disease and can easily cause transduction in many
tissues of CNS. Retroviral vectors are also easily prepared & can target specific
cells but there is limitation that they cause mutagenesis. Adenoviruses &
herpes viruses are also used but more research is needed to improve them.
R. M., july, 2015)
Figure: Genetic
therapy for the nervous system (Hum Mol Genet et.al 2011).
Routes of gene delivery to the CNS. CED of viral vectors into the brain
improves considerably their distribution in target structures and hence
transduction volumes. This technique can yield volumes of transduced cell
distribution 3–3.5-fold larger than the infused volume, which is highly
significant for human applications. Viral vectors or secreted transgene
products (growth factors, lysosomal enzymes) can be further distributed from
the primary target structure by axonal transport (top left diagram). Infusion
of recombinant proteins or oligonucleotides into the brain ventricular system,
or intrathecal space, leads to widespread CNS distribution via CSF flow. An
alternative strategy is to use viral vectors to engineer ependymal cells lining
the ventricles or choroid plexus cells to secrete therapeutic proteins into CSF
(top right diagram). The BBB with its many constituents has thwarted most gene
transfer vehicles from entering the brain from the vasculature. In recent
years, PILs and a new generation of viral vectors (AAV and SV40) have been
shown to mediate efficient CNS gene transfer after i.v. infusion in newborn and
adult animals (bottom right diagram).
The vectors are inserted into the brain by direct
injection into parenchyma tissue of brain. It is done to target the diseased
structure of brain. The vectors can also be entered into CNS via CSF in lateral
ventricles or intrathecal spaces. Ideal route of delivery of vectors is through
vasculature (Steven et.al 2011).
Figure: Genetic therapy
for the nervous system (Hum Mol Genet et.al 2011).
Schematic
representation of existing cellular therapies, including iPS cells, which
require vector-based strategies to generate neuroprecursor cells and neurons to
ultimately treat neurodegenerative diseases. A variety of cellular therapies
have been devised to repair and replace degenerated neuronal networks within
the CNS. These strategies utilize multiple sources of neurons and
neuroprecursor cells, including fetal brain, pre-implantation embryos, mesenchymal
stem cells and iPS cells. The generation of iPS cells requires the use of viral
vectors to deliver multiple genes key for the molecular reprogramming of
patient fibroblasts. These iPS cells can be differentiated into neurons or
neuroprecursor cells that are subsequently transplanted into the diseased
brain.
Primary metabolic disorders are cured by direct
delivery of vectors in brain while secondary metabolic disorders require
continuous expression of genes.
are also used in gene therapy. These are Plasmid DNA Nano particles. As naked
DNA is unstable, so it is not directly injected into brain parenchyma. Instead
it is packed into Nano particles. These are simple, cheap an nontoxic.
R. M., july, 2015)
Organically modified silica based nanoparticles are used to pack the desired genes.
Since the idea of Gene Therapy was given many
challenges have been faced by researchers. Continued trials have also made
improvements in Gene Therapy procedures.
methods to modify the nervous system. These methods include delivery of genes
to CNS, Sequence-targeted regulatory molecules & genetically modified
cells.
disease by replacing or correcting defective genes. This approach is used in
recessive disorders in which defective genes are substituted by correct ones.
Some diseases are caused by defective proteins. These are treated by simply
suppressing the expression of their genes into proteins. In many diseases of
CNS factors are introduced which cause the symptoms of the disease to be
delayed or suppressed.
Related
Work:
Use
of Vectors in curing Neurological Diseases:
AAV vectors are being used in clinical trials to
treat Alzheimer’s disease, Parkinson’s Disease, Epilepsy, Amyotrophic Lateral Sclerosis
(ALS). Among AAV Vectors Especially serotype 2 (AAV2) is used. Retroviral
Vectors are second most commonly used vectors especially in ex vivo
transduction of cells to express nerve growth factor (NGF). Lentiviral Vectors
are used in clinical trials to treat Parkinson’s disease.
Lysosomal
Storage Diseases:
These are storage diseases which are caused due
deficiency of lysosomal enzymes. These are monogenic diseases. Successful clinical
trials have been done on animals for the cure of these diseases.
Two approaches have been used in animal models.
i. 1. Intraparenchymal infusion of recombinant
viral vectors in brain cells.
ii 2. Successful Bone marrow transplantations in
mouse models with ex vivo lentivirus vector modified-autologous (HSCs). These
enzymes are wide spread when AAV-mediated modifications are carried out in
structures such as cerebella nuclei, ventral tegmental area or thalamus.
Now these are tested on human in clinical trials for
the treatment of lysosomal storage diseases.
Alzheimer’s
disease & other Amyloidopathies :
It is a neurodegenerative disorder in which memory
loss occurs and there is no cure of it.
Tuszynski & colleagues delivered nerve growth
factor (NGF) prototypical neurotrophin in rodents and non-human primates using
retrovirus vector-transduced fibroblast grafts. The result was positive and
restored cholinergic neurons in these experimental models. Phase I clinical
trials were carried out using ex-vivo NGF gene therapy. It was very successful.
Ceregene carried out another gene therapy approach
in which phase I & II clinical trials were carried out using AAV vectors to
produce NGF.
Spinal
Muscular Atrophy (SMA):
It is an autosomal recessive disease caused by loss
of function of motor neuron gene SMN1. It leads to degeneration of motors
neurons & mortality of infants. Gene Therapy approach used replacement of
the missing gene with intravenous administration of AAV9 vector. It delivered
SMN1 cDNA to spinal cord in mouse model. It resulted in correction of motor
function & survival of mouse.
Amyotrophic
Lateral Sclerosis (ALS):
It is a muscle weakness resulting from loss of motor
neurons in brain & spinal cord. It is caused by mutations in gens which
produce proteins with toxic functions. When growth factor was introduced in
mouse model it increased the survival but did not end the disease. It delayed
the symptoms of disease but the disease continued its progression.
Brain
Tumors:
Glioma Tumors i.e glioblastoma GBM is the most
common adult brain tumor which is untreatable. It is invasive in brain brain
tissue. Therapies have been done on experimental models of brain to eliminate
tumor mass and inject virus vectors to kill remaining tumor cells.
Following strategies were used to treat brain tumor.
i.
Prodrug
Activation:
Viral
vectors are introduced which produce enzyme to activate prodrugs into active chemotherapeutic
agents in the tumor to cure it.
ii.
Viral
Oncolysis:
In this strategy mutant HSV-1 and
adenoviruses are introduced which carry therapeutic genes.
iii.
Cellular
Delivery:
In this strategy neural stem cells
and mesenchymal cells are used to deliver toxic agents to tumor cells.
iv.
Immunotherapy
to Target Tumor Antigens:
It is used to make immune system
strong. Antigen EGFRvIII is introduced as vaccination with oncolytic HSV
vectors & cyclophosphamide.
v.
Zone
of Resistance:
This increases the resistance of
brain to tumor. AAV vectors in brain produce interferon-beta which suppress
growth of tumor.
Gene
Therapy in treatment of Chronic Pain:
Chronic pain have affected about 80 million
Americans in their life. It is caused by inflammation or by damage caused to
nerve. Gene therapy has been used to treat chronic pain at the level of spinal
cord. Phase I human trial was carried out on cancer patients having pain from
bone metastasis. Herpes Simplex Viral Vector was used which carried human
pre-proenkephalin (hPPE) gene which produced opiod peptides met- &
lew-eukephalin (ENK). This showed positive response in relieving pain in
patients for up to 1 month after injection. (10) Dr. Tao group showed that
restoring kv,2 expression lessen the chronic pain in sensory neurons.
Figure Nerve injury-induced Kv1.2 downregulation
triggered by myeloid zinc finger protein 1 (MZF1)-mediated Kv1.2 antisense (AS)
RNA expression in the injured dorsal root ganglion (DRG). (A) Under normal
conditions, Kv1.2 mRNA that is transcribed from the genome is translated into
Kv1.2 protein, resulting in normal expression of Kv1.2 channel at DRG neuronal
membrane. (B) Under neuropathic pain conditions, peripheral nerve injury
promotes the expression of the transcription factor MZF1 in DRG. The increased
MZF1 binds to the promoter region of Kv1.2 AS RNA gene and triggers its
expression. The latter specifically and selectively inhibits the expression of
Kv1.2 mRNA via extensive overlap of their complementary regions, leading to a
reduction in the membrane expression of Kv1.2 only, not other Kv subunits
(e.g., Kv1.1), in the DRG neurons.
Adeno-associated virus (AAV)
mediated transfer of Kv1.2 sense RNA for the reduction of DRG neuronal
excitability. (A) Before AAV injection into the DRG
of rats with peripheral nerve injury, a nerve injury-induced increase in DRG
Kv1.2 AS RNA triggered by MZF1 knocks down expression of Kv1.2 mRNA and
protein, resulting in an increase in DRG neuronal excitability under
neuropathic pain conditions. (B) After AAV injection
into the DRG of rats with peripheral nerve injury, AAV mediated transfer of
full length Kv1.2 sense (SE) RNA rescues nerve injury-induced DRG Kv1.2
downregulation at the DRG neuronal membrane through not only its direct
translation into Kv1.2 protein but also its indirect blockage of nerve
injury-induced increase in Kv1.2 AS RNA expression via extensive overlap of
their complementary regions. AAV mediated transfer of Kv1.2 SE RNA fragment
(-311 to +40) also rescues nerve injury-induced DRG Kv1.2 downregulation through
its blockage of nerve injury-induced increase in Kv1.2 AS RNA expression via
partial overlap of their complementary regions, although this RNA fragment
cannot be translated into Kv1.2 protein. Maintaining normal Kv1.2 expression at
DRG neuronal membrane reduces nerve injury-induced neuronal hyperexcitability
at DRG neurons and consequently decreases spinal central sensitization,
resulting in neuropathic relief.
Rett
Syndrome:
In Rett Syndrome, slowing of development occurs such
as slow brain & head growthloss of hand movements & use of hands.
Problems with walking & intellectual disability also occur. Rett syndrome
has been treated with gene therapy.MeCP2 gene is inserted in neurons &
glial cells which resulted in lessening the disease symptoms.
R. M., july, 2015)
Sandhoff
Disease:
It is caused due to mutation of gene which causes
accumulation of GM2 in CNS. AAV mediated gene therapy is used to treat this
disease. Successful clinical trials have been done on in murine and cat brain
models. It showed improvements and hopeful for treating the disease in humans.
Rockwell1, 2015)
Discussion:
Gene Therapy is the most advance treatment strategy
used to treat many neurological disorders in clinical field. Two decades back
it was impossible to treat neurological disorders. They were considered as
inherited and genetic disorders. It was impossible to cure genetic disorders.
But in the advancement of genetics it has become possible to even cure there
genetic disorders. Researchers first made clinical trials on animal models.
Within these 20 years gene therapy has become practically possible. Now many
neurological disorders are being treated by using gene therapy. Different
strategies were implied such as to replace a defected gene or to introduce new
gene into the CNS. Researchers faced many problems in the development of
vectors. But their continued efforts made possible to make a variety of vectors
to be used as gene carriers to the brain. Now a days work is being done on
producing non-viral vectors. Routes of
administration were also considered a problem in the past but now there are
many routes by which genes are inserted in CNS. In other words, many efforts
were made by researchers to develop better gene therapy techniques.
Future
Perspectives:
A lot of scope is remaining in this field. There is
a need of a lot of work in the area of gene therapy. More research should be
done to make the process more better and safe. Especially in the field of
producing non-viral vectors. A lot of effot is required in this field to
produce efficient an safe vectors. More drugs should be made so that the
treatment becomes common. There is still need for more research in the field of
making genetic drugs for common people.
Conclusion:
In Short, many clinical trials have been made in the
field of gene therapy to cure CNS disorders for the past 20 years. A lot of
improvement has also been made in this field but there is still need of more
improvement in this regard.
The idea of using gene therapy is ideal for curing
CNS disorders than using traditional medicines but still more research is
needed in this field. A breakthrough is still needed for the treatment become
common Hence, it is concluded that more work should be done in the field of
gene therapy
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About Author:
Mr. Imran Zafar
has completed his Bachelor of Science (BS) degree in Bioinformatics from
COMSATS Institute of Information Technology Islamabad Sahiwal campus under
supervision of Dr. Ahmad Ali and Master of Science (MS) in Bioinformatics from
Department of Bioinformatics and Computational Biology, Virtual University of
Pakistan, Lahore, Punjab, Pakistan under supervision of Dr. Muhammad Tariq
Pervez. For research work during BS and MS he has also done internships from
School of biological Science (SBS), University of Veterinary and Animal Sciences
(UVAS) and Center of Excellence in molecular biology (CEMB) Lahore. He has published
several research articles and book computers in reputed journals recognized from
Higher Education Commission (HEC) of Pakistan.
His research is mainly focused on the field of Bioinformatics, Genomics,
Computational Biology and Molecular Biology in the domain of life science to
performed computational analysis. He is now working in Ministry of Education as
a Science subject instructor in the Department of Education Punjab,
Pakistan.