NTCC SEMINAR REPORT

GENE THERAPY FOR NEURODEGENERATIVE

DISORDERS

Submitted in partial fulfilment of

B.TECH BIOTECHNOLOGY

by
Shreeya Sharma
B.Tech Biotechnology (2017-2021)
Enrollment no. A0504117075
Roll no. BTB/17/156

Under the supervision of

Dr Maansi Vermani
Associate Professor, Amity Institute of Biotechnology

At
Amity Institute of Biotechnology
Amity University, Uttar Pradesh
Sector 125 Noida, Uttar Pradesh, India – 201303
September 2020
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AMITY INSTITUTE OF BIOTECHNOLOGY

SEMINAR REPORT - 2020

Ref No.-AIB/20……/____________

PROJECT TITLE : GENE THERAPY FOR NEURODEGENERATIVE DISORDERS

COURSE : B.TECH BIOTECHNOLOGY

SEMESTER : 7

NAME OF STUDENT : SHREEYA SHARMA

ENROLLMENT NO : A0504117075

BATCH : 2017-2021

TRAINING PERIOD : SEPTEMER 1 – SEPTEMBER 30 2020

NAME OF EXTERNAL GUIDE: N/A

NAME OF INTERNAL FACULTY COORDINATOR (IFC): DR. MAANSI VERMANI

_____________________________________________________________________________________________________
J- 3 Block, Amity University Campus, Sector – 125, NOIDA – Greater NOIDA Expressway,
NOIDA – 201 313, Gautam Buddha Nagar(Uttar Pradesh, India) Tel. No. - +91 120
4392195, Fax: 0120- 4392947
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AMITY INSTITUTE OF BIOTECHNOLOGY

SEMINAR REPORT-2020

Ref No.-AIB/20…./____________ Date

CERTIFICATE
This is to certify that the NTCC SEMINAR Report entitled GENE THERAPY FOR
NEURODEGENERATIVE DISORDERS submitted to Amity Institute of Biotechnology,
Amity University, Uttar Pradesh carried out in partial fulfillment for the award of B.TECH
BIOTECHNOLOGY Degree is a bonafide work carried out by Ms. SHREEYA SHARMA
Enrolment No A0504117075 of semester 7 from 1ST SEPTEMBER 2020 to 30TH
SEPTEMBER 2020 time period.

No part of this project work has been produced elsewhere for any degree.

DR. MAANSI VERMANI

Internal Faculty Coordinator (IFC)
(Name and Signature)

_____________________________________________________________________________________________________
J- 3 Block, Amity University Campus, Sector – 125, NOIDA – Greater NOIDA Expressway,
NOIDA – 201 313, Gautam Buddha Nagar(Uttar Pradesh, India) Tel. No. - +91 120
4392195, Fax: 0120- 4392947
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Table of Contents
1. INTRODUCTION................................................................................................................1

2. DELIVERY VEHICLE FOR GENE THERAPY.............................................................3

2.1 Viral Vectors........................................................................................................................3

Adeno-associated Virus (AAV).............................................................................................3

Lentiviruses and Retroviruses................................................................................................4

Adenoviruses (Adv)...............................................................................................................4

2.2 Non-Viral Vectors................................................................................................................4

3. TARGET SELECTION FOR NEURODEGENERATIVE DISORDERS.....................6

3.1 Endoplasmic reticulum stress and unfolded protein response.............................................6

3.2 mTOR signalling..................................................................................................................6

3.3 Mitochondrial function.........................................................................................................7

3.4 Epigenetic regulation...........................................................................................................7

3.5 Autophagy............................................................................................................................8

4. FUNDAMENTAL MODES OF GENE THERAPY IN THE CNS.................................9

4.1 Gene Overexpression...........................................................................................................9

4.2 Gene silencing......................................................................................................................9

4.3 Gene editing.......................................................................................................................10

5. GENE DELIVERY ROUTES IN THE CNS...................................................................11

5.1 Intravenous injection..........................................................................................................11

5.2 Intraparenchymal injection.................................................................................................11

5.3 Intrathecal, intracisternal, and intracerebroventricular injections......................................11

5.4 Ex-vivo gene therapy.........................................................................................................12

6. RECENT AND ONGOING CLINICAL TRIALS..........................................................13

6.1 Parkinson’s disease............................................................................................................13

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6.2 Alzheimer’s disease...........................................................................................................13

6.3 Amyotrophic Lateral Sclerosis (ALS)...............................................................................14

CONCLUSION.......................................................................................................................16

REFERENCES.......................................................................................................................17
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ABSTRACT
Gene therapy has made important advances over the last decade. Among neurological
diseases, severe genetic neurodegenerative conditions have been the focus of initial clinical
applications. Gene therapy has also addressed complex neurodegenerative diseases,
particularly Parkinson’s disease, with encouraging results in human patients, demonstrating
that specific targeting of central nervous system (CNS) cells is a relevant strategy for severe
pathologies and that efficient access to the CNS with viral vectors is an achievable goal.
Efforts to improve outcomes are focusing on three main areas: vector design and the
identification of new vector serotypes, mode of delivery of gene therapies, and identification
of new therapeutic targets. This seminar report attempts to summarize the gene therapy
clinical applications that have been conducted for neurodegenerative diseases. Limitations
and hurdles to obtain and demonstrate benefit in patients, and the new developments that
should allow new clinical applications with high beneficial potential.
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1. INTRODUCTION
Gene Therapy is the process of using genes for the treatment and prevention of diseases that
are not curable by the conventional methods. The treatment is achieved by insertion of a gene
or a group of genes into a patient’s cells. This genetic material will provide a therapeutic gene
or gene products that will assist in permanent restoration of the missing function of the
patient’s cells. There are several approaches to gene therapy like, replacing a mutated gene
with a healthy copy of the gene, or inactivation (“knock-out”) of a mutated gene, or
introducing a therapeutic gene that will help fight the disease. Gene Therapy may be classical
or non-classical. In classical gene therapy, genes are introduced to the target cells to obtain
expression of the introduced genes. In non-classical gene therapy on the other hand, the focus
is on inhibiting the expression of the genes responsible for the disease, or to correct defective
genes and restoring their normal expression.

Diseases of the Central Nervous System (CNS) have posed multiple hurdles and limitation in
the conventional pharmacological approaches of their treatment. For instance, the brain is a
complex organ and brain disease processes have a spectrum of pathological states. These can
have long-lasting consequences on a person’s neural development, brain function, plasticity
and metabolism. These neurodegenerative disorders remain largely ununderstood or limitedly
understood, which limits therapeutic advances.

Another limitation is the many barriers protecting the brain which limit the access to the
organ. The Blood Brain Barrier prevents the delivery of any therapeutic agents to the central
nervous system. These physical constraints are the reason why most drugs and neurosurgeries
are not effective in the treatment of neurological disorders.

Additionally, the complexity and the limited accessibility of the organ leads to difficulty in
the evaluation of the clinical outcome after the treatment. Neurodegenerative disorders do not
have a linear progression. Therefore, a history of the disease is important to understand the
outcome of the treatment. This makes clinical trials, technologies like brain imaging (MRI,
DTI, PET), and surrogate markers highly important for proof-of-concept studies of
neurodegenerative disorders.
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Several brain diseases have been led to clinical trials, particularly genetic neurodegenerative
disorders. Over the past few decades these researches and clinical trials have made huge leaps
in our understanding of these disorders. Increased understanding of the pathological
mechanisms of these diseases has led to advances in therapeutic technologies, including
identification of novel targets and vectors. A considerable number of clinical trials have been
performed over the years. The early clinical trials had a high failure rate in achieving
satisfactory therapeutic effects. However, with improvements in vectors and delivery
systems, gene therapies have shown higher therapeutic safety. Gene therapies have been
found to give highly functional clinical outcomes in experimental models of many
neurodegenerative disorders, including Alzheimer’s disease (AD), Huntington disease (HD),
aromatic-L-amino-acid decarboxylase (AADC) deficiency, and Parkinson’s disease.
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2. DELIVERY VEHICLE FOR GENE THERAPY

While developing a gene therapy, researchers have to take many factors in consideration.
This includes appropriately designing and executing the experiment, choosing the most
suitable delivery vehicles and methods, and deciding what expression system to use.

Choosing the delivery vehicle is the first step a researcher needs to take while designing a
gene therapy. A lot of factors have to be taken into consideration, including the amount of
nucleotides the genetic material encompasses, the phenotype of the target cell, the aim of the
experiment (for example, therapeutic gene), and the available laboratory resources.

2.1 Viral Vectors

Recombinant viral vectors are a popular choice for delivery of genetic material into a host
animal. These recombinant viral vectors take advantage of the virus’ ability to infect a host
while removing the virus’ ability to replicate itself or to cause damage to the host cell. The
three most commonly used viral vectors are adeno-associated virus (AAV), lentivirus (LV) or
retrovirus, and adenovirus (Ad).

Adeno-associated Virus (AAV)

Adeno-associated viruses (AAV) have strong transduction profiles and have been found to
be quite safe in both animals and humans. Therefore, AAV are the most commonly used gene
therapy vector in the nervous system. The recombinant AAV (rAAV) contains only two
genetic elements that correspond to the wildtype: the two inverted terminal repeats (ITRs),
responsible for the packaging of the genome in the viral capsid. A high number of AAv
serotypes have been created since its discovery, each with a different capsid surface. More
than 100 AAV variants consisting of 13 serotypes (AAV 1-13) have been identified in
humans and non-human primates. AAV2 is the most popular variant of AAV for gene
therapy of neurodegenerative disorders, and has been used in numerous clinical trials. AAV2-
NGF has shown evidence of treating the cognitive decline in Alzheimer’s Disease (AD) and
related dementia. The variants AAV9 and AAVrh10 have been found to effectively penetrate
the blood brain barrier, making them important vectors. One of the drawbacks of rAAV is its
small size, that limits its transgene capacity. Any attempt to more than 5kb of genetic
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material often leads to lower packaging efficiency and reduced titres. Another limitation to
rAAV is that the packaging is labour intensive and requires specialised equipment.

Lentiviruses and Retroviruses

Retroviruses and their subfamily, Lentiviruses, have the ability to integrate in the host cell’s
genome and thus their genomes do not get diluted with cell division. The non-lentivirus
retroviruses cannot transverse the nuclear membrane, and thus are limited to infecting
actively dividing cells. Lentiviruses, on the other hand, can easily get imported into the
nucleus and can infect both dividing and non-dividing cells. These viruses can fully integrate
in the host genome through reverse transcription. This provides a stable and longer transgene
expression. This family of viruses has a larger size and this allows the inclusion of roughly 9
kb of genetic material.

Adenoviruses (Adv)

Adenovirus is an icosahedral capsid virus that cannot insert its own gene in the host genome.
This leads to an excellent safety profile, even though the transgene expression is relatively
transient. The rAd has the capacity to carry roughly 35 kb of genetic material, which makes it
a desirable delivery vehicle of larger genes.

2.2 Non-Viral Vectors

Although viral vectors are a popular choice for delivery vehicles, they have many drawbacks,
including broad tropism, limited loading capacity, difficulty in vector production, and host
inflammatory responses. These drawbacks can be avoided by using non-viral vectors. Non-
viral vectors can be classified into lipid-based vectors and polymeric vectors. Lipid-based
vectors are the most extensively used non-viral vectors. Cationic lipids, like DOTMA, DC-
cholesterol, DOTAP and DSPE, are popular choices of vectors for gene therapy. They have
three main domains: a hydrophobic tail, cationic cap, and linking groups. However, cationic
lipids have nonspecific binding and rapid clearance, which cause an unsatisfactory
pharmacokinetic biodistribution. Apart from these lipids, lipoids (lipid-like materials),
magnetic nanoparticles and exosomes have been experimentally proven to be promising gene
delivery vehicles for neurodegenerative disorders. For example, magnetic ferric oxide
nanoparticles coated with N-isopropylacrylamide derivatives can significantly alleviate
Parkinson’s Disease (PD) in mice. Cationic polymers are another type of non-viral vectors
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that are an attractive delivery vehicle for neurodegenerative disorders. They have the capacity
for endosomal escape, as a result of their spherical architecture, sponge-proton effect and
chemical diversity. Non-viral vectors have improved significantly in the recent years and
further research into these is likely to advance the treatment of neurodegenerative disorders.
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3. TARGET SELECTION FOR

NEURODEGENERATIVE DISORDERS
Neurodegenerative disorders cause progressive dysfunction of neurons in specific regions of
the Central Nervous System, and eventually lead to disability and/or death. It is necessary to
identify pathological targets for gene therapy, as the available therapeutic agents offer only
symptomatic relief.

3.1 Endoplasmic reticulum stress and unfolded protein response

Almost all neurodegenerative disorders show an abnormal accumulation of misfolded
proteins. These misfolded proteins get aggregated and cause endoplasmic reticulum stress
and ER-associated degeneration. Amyloid β oligomers and α-synuclein are two misfolded
proteins that get aggregated in the lumen of the ER. Here they destabilize the calcium
homeostasis and distort the unfolded protein response (UPR) signalling. The UPR signalling
is important for restoration of cellular proteostasis, but its distortion leads to proapoptotic
responses and neuron death. Researchers have suggested that gene therapies targeting the
UPR signalling and improving protein folding and reducing the ER stress, will provide long
term therapeutic effects. Gene therapy using overexpression of BiP (glucose regulated protein
78) to treat experimental Parkinson’s Disease has been able to reduce dopaminergic neuron
apoptosis, enhance motor performance and delay disease progression. Intracerebral delivery
of AAV6-SIL1 to mouse models of Amyotrophic Lateral Sclerosis (ALS) has been found to
restore ER homeostasis and prolong survival. This approach hasn’t been used in clinical trials
yet since safety assessment in long term follow up studies is still required.

3.2 mTOR signalling

mTOR (Signalling transductions of mammalian targets of rapamycin) have been found to
play a pathological role in many neurodegenerative disorders, including Alzheimer’s,
Parkinson’s, Huntington’s disease, traumatic brain injury and optic nerve injury. Abnormal
mTOR signalling likely leads to the degeneration of neural cells since no clear proof of toxic
protein accumulation has been found. Experiments with a mice model of Parkinson’s Disease
have found that AAV-based overexpression of S6K1 and AKT has therapeutic effects.
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Several studies have reported hyperactivated mTOR signalling in Huntington’s disease and
Alzheimer’s disease, and that reinstating aberrant mTORC1 activity can restore neural
activity.

3.3 Mitochondrial function

Mitochondrial respiratory dysfunction contributes to many neurodegenerative disorders, like
Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, glaucoma, ALS and
lysosomal storage disease. All these diseases and disorders show signs of mitochondrial
respiratory dysfunction, including oxidative damage, limited regulation of mitochondrial
quality, NAD+ depletion, unbalanced mitochondrial calcium homeostasis, disrupted ATP
synthesis and protein aggregates. Therapeutic agents that promote mitochondrial regeneration
or inhibit mitochondrial disruption have been tested on mice models. These agents like
Bendavia, CoQ, MitoQ, help rescue neurodegeneration. Studies on experimental Parkinson’s
disease and Huntington’s disease have found that gene therapies that overexpress regulators
of mitochondrial oxidative stress and dynamics can reduce neurocytoxity. Clinical trials of
these mitochondrial treatments haven’t been as satisfactory as researchers may have hoped.
This may be because by the time patients enter clinical trials, their disorders are too advanced
for effective treatment.

3.4 Epigenetic regulation

Epigenetic regulation is important for axonal development and neuronal survival. Epigenetic
regulation consists of many mechanisms like histone variant, DNA methylation, chromatin
remodelling, and histone post-translational modification. Studies have found that changes in
H3K27ac and H3K5me3 occur in genetic variants of Alzheimer’s disease, and the protein
H4K16ac, which is involved in DNA repair, is significantly reduced in the cortex of AD
patients. Multiple reports have associated the loss of the protein H3K4me3, which is
responsible for gene activation, with Parkinson’s disease and Huntington’s disease. However,
the overexpression of this protein has been found to be accelerate A-T mutation that mitigates
behavioural impairments and neurodegeneration. HDAC inhibitors have been shown to
prevent neurodegeneration in models of glaucoma, AD and Huntington’s disease. These
studies show that epigenetic regulation is involved in neurodegenerative disorders and better
understanding of these mechanisms is important for development of epigenome targeted
therapies.
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3.5 Autophagy
Autophagy is the process by which evolutionarily conserved intracellular machinery degrades
dysfunctional organelles and denatured proteins in lysosomes. Autophagy removes misfolded
proteins, including tau, HTT and α-synuclein. Autophagy has been found to be associated
with some neurodegenerative disorders like Alzheimer’s, Parkinson’s, Huntington’s diseases,
and ALS. Studies have indicated that PTEN-induced overexpression of putative kinase 1
clearance of dysfunctional mediated by AAV2 promotes autophagy that facilitates clearance
of dysfunctional mitochondria in experimental Alzheimer’s disease. The clearance of
mitochondrial dysfunction leads to amelioration of loss of mitochondrial functions and
cognitive decline. Similarly, overexpression of transcriptional factor EB (TFEB) via
intracerebral injection of AAV vectors can effectively alleviate α-synuclein induced
neurodegeneration in Parkinson’s disease. Additionally, overexpression of AAV9 snapin
reduces the defects in retrograde transport, which improves mitochondrial function and
enhances motor neuron survival and mitigates disease phenotypes in mouse models of ALS.
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4. FUNDAMENTAL MODES OF GENE THERAPY IN

THE CNS
4.1 Gene Overexpression
When the intended effect in a CNS gene therapy is to (ectopically) overproduce a certain
protein, transgene overexpression is a common approach. This protein may be used to elicit a
therapeutic or symptomatic effect, or it may be used to model a disease by producing a toxic
effect. For example, in Parkinson’s disease, the overexpression of members of glial cell line-
derived neurotrophic factor GNDF are currently being clinically tested for their efficacy to
halt neurodegeneration. Their preclinical testing using rAAV and LV vectors have proven
their strong neurotrophic potential but their effect is mediated by transmembrane signalling.
Thus, the protein has to be released in the surrounding area. This is done by including a
signal peptide targeting the protein for release. Here the phenotype of the target cells is not as
important as their anatomical location. Some other approaches have transgenes that activate
inside specific target cells only. The protein α-synuclein plays an important role in
neurodegeneration of SNc neurons and gene therapy mediated overexpression of this protein
in specifically nigral neurons is a common model.

4.2 Gene silencing

Many neurodegenerative disorders are caused by mutations causing a toxic increase in
function of the gene. For example, Huntington’s disease is caused by a CAG expansion in the
gene encoding the protein huntingtin. A gene therapy for this disease has been using RNA
silencing techniques like short hairpin RNA (shRNA) or MIR. The preclinical studies have
shown encouraging effects on the disease progression after the reduction in expression of
mutant huntingtin. However, this method has some disadvantages. Both shRNA and MIR
cause the inhibition of nuclear export due to increase in quantity of exportin-5. This can lead
to off-target toxicity. Thus, any experiment involving shRNAs or MIRs should be controlled
properly.

An important consideration while creating knockout models is the target gene itself. Proteins
like huntingtin are involved in the normal cellular processes too and may be crucial for the
survival of the cell. Therefore, high amount of reduction in their mRNA may produce some
 xv

negative effects. An approach to combat this problem is the “kill and replace” approach. In
this, a ‘hardened’ copy of the wild cDNA of the target is also inserted along with the shRNA.
This hardened copy has many silent mutations that makes it immune to any effect by shRNA
or MIR.

4.3 Gene editing

In gene editing, a vector-based genome editing is employed to remove or replace the3 toxic
gene. Recent researches have helped create engineered nucleases that bind with a specific
sequence of the host genome and cause a double-stranded break. This activates the cell’s
DNA repair mechanism and the toxic gene can be easily removed. In viral vectors, zinc
finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats
(CRISPR) systems are best suited and most commonly used. However, in non-viral delivery,
transcription activator-like effector nucleases (TALENs) are used.
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5. GENE DELIVERY ROUTES IN THE CNS

Delivery of the gene to the CNS, sensory organs and the eyes is a challenge and a proper
balance has to be attained between treatment efficiency and safety. This balance is achieved
by the judicious combination of delivery routes and vectors.

5.1 Intravenous injection

The physiological barriers, like the blood brain barrier, that protect the CNS tissues are
serious hurdles to gene delivery. AAV9 vector can easily penetrate these barriers after an
intravenous injection. This helps in a widespread CND distribution and expression that can
treat multifocal disorders effectively. Some other AAV variants that can do this task are
AAV-AS, AAV-B1, ANF AAV.PHP.B. One potential disadvantage of intravenous delivery
is the delivery of the genes to other body tissues. Some major obstacles to this delivery
method are the requirement of large doses, the potential generation of antibodies against the
virus and some safety concerns.

5.2 Intraparenchymal injection

Intraparenchymal injections provide local delivery of the genes directly into the neurons and
the region of interest and has very little biodistribution in the peripheral organs. Vectors that
can bind themselves to the heparin sulfate proteoglycans (HSPGs), like AAV2, AAV-DJ88,
and AAV6, do not diffuse into the peripheral regions, unlike the vectors that do not bind to
HSPGs, like AAV1, AAV8, AAV9 and AAVrh10. AAV2 provides an appropriate vehicle to
limit diffusion in Canavan disease and in Parkinson’s disease. AAV2-ASPA has been used to
treat Canavan, while AAV2-AADC has treated Parkinson’s disease.

5.3 Intrathecal, intracisternal, and intracerebroventricular

injections
Gene delivery can also be done by administration into various cerebrospinal fluid
compartments. Intrathecal injections of AAVs help deliver vectors to sensory neurons in
dorsal ganglia or motor neurons. This has been well received by the host bodies in many
preclinical studies. AAVs like AAV9 and AAVrh10 target spinal cord motor neurons after
intrathecal injection in non-human primates. Preclinical studies with intracisternal and
 xvii

intracerebroventricular injections have shown transgene expression in spinal cord and

cerebral tissues. This has alleviated symptoms in models of Alzheimer’s disease, ALS, and
spinal muscular atrophy.

5.4 Ex-vivo gene therapy

This gene therapy approach utilizes manipulation of haematopoietic stem cells, obtained from
the blood or bone marrow, with LVs. This is followed by autologous transplantation of
transduced cells. This causes a renewal in the transgene therapeutic proteins after
haematopoietic reconstruction. This is especially useful in treating the disorders where
microglial defect is the primary lesion, like X-linked adrenoleukodystrophy (X-ALD). This is
also used to deliver secreted proteins to the brain and using the microglia as a cell factory.

Figure 1: In vivo and ex vivo gene therapy strategies for the treatment of central nervous system (CNS)
diseases. (Clinical Gene Therapy for Neurodegenerative Diseases, Francoise Piguet, Sandro Alve)
 xviii

6. RECENT AND ONGOING CLINICAL TRIALS

In recent years, several researches and clinical trials have been started to combat
neurodegenerative disorders. These clinical trials utilise and are based on the different
delivery vehicles and mechanisms discussed in this paper.

6.1 Parkinson’s disease

PD is characterised by loss of dopaminergic neurons of the substantia nigra pars compacta
(SNc) and reduction in levels of dopamine in the striatum. Symptoms include rigidity, resting
tremors, and motor function impairment. The possibility of using gene therapy for treating
PD has been extensively studied and several gene therapies have been developed. However,
these have mostly failed the Phase II trials where the majority have failed to show any
improvement beyond placebo effect.

The only trial that succeeded in the Phase II of the trials was the delivery of AAV2-glutamic
acid carboxylase (GAD) by direct injection into the subthalmic nucleus. The patients who
received AAV2-GAD showed improvement in symptoms as compared to control patients.

In another trial, six patients with moderate PD were administered AAV2 with neuturin
(CERE-120) in both the putamen and substantia nigra. This was done to counter the deficits
in the axonal transport, which limits the efficacy of the neurotrophic factors. The results
showed tolerance and absence of adverse effects, indicating that this gene therapy was
feasible, safe and well tolerated in PD patients. Long-term follow-up of the patients showed
that the motor capabilities remained stable and sometimes even slightly improved. This
provided evidence that AAV2-meuturin is safe in the long term as well.

Three enzymes, tyrosine hydroxylase, guanosine triphosphate cycohydrolase I, and AADC,

are involved in the production of dopamine from L-tyrosine. Preclinical studies in animals
involving the three enzymes using viral vectors showed improvement in PD phenotype.

6.2 Alzheimer’s disease

AD is a progressive neurodegenerative disorder. It is characterised by problems with
memory, thinking and behaviour. It is caused by degeneration of cholinergic neurons and
 xix

build-up of amyloid β plaques. Familial AD results from mutations in amyloid β precursor

proteins (APP) gene, or in the proteases that cleave the precursor protein into fragments.

The only gene therapy clinical trials that have been launched for AD were based on
intracerebral delivery of AAV2 encoding nerve growth factor (NGF). The therapy was well
tolerated, with follow-up data for up to 2 years. Analysis of post-mortem tissue identified
expression of NGF in an active form. However, the Phase II trial failed to meet its primary
endpoints.

One strategy for inhibiting plaque formation is to decrease levels of Ab and tau in the brain.
An AAV expressing a miRNA to knockdown acyl-CoA cholesterol acyltransferase 1
(ACAT1) was shown to reduce Ab levels in a triple transgenic mouse model of AD A
multicentric Phase II is ongoing on 49 patients, but results are not yet available.

6.3 Amyotrophic Lateral Sclerosis (ALS)

ALS (also known as Lou Gehrig’s disease) is a progressive, fatal neurodegenerative disease.
It results from a loss of both upper and lower motor neurons in the brain, brainstem, and
spinal cord. It is usually fatal within 2–5 years. ALS can be either sporadic or familial in
origin, and a variety of genes are linked to disease development.

For familial ALS, altering the expression of the affected gene has proven effective. This
approach has primarily been tested for the superoxide dismutase 1 (SOD1) gene because
mutations in SOD1 were among the first to be identified as causing ALS. Delivering
antisense oligonucleotides (ASOs) to SOD1 intrathecally progressed to a Phase I trial, with
some patients receiving repeat treatments. Efficacy was not expected, as most patients
received a single dose, but the safety profile of this treatment was established. One
disadvantage of administering ASOs on their own is the need for constant infusion or repeat
dosing. Using a viral vector to deliver an ASO or a short hairpin (sh) RNA circumvents this
issue. Two proof-of-principle studies have used AAV9-SOD1-shRNA to knock down SOD1
gene expression in rat models.

For sporadic ALS, a more general neuroprotective approach needs to be adopted. One
method of achieving this is by delivering growth factors to support the motor neurons.
 xx

Growth factor delivery has shown promise in preclinical testing but little efficacy in clinical
trials. However, there is potential for therapeutic efficacy from growth factors. Vascular
endothelial growth factor (VEGF) was linked to ALS when mice with a deletion in the VEGF
promoter region developed motor neuron disease similar to SOD1 mice. Gene delivery
experiments resulting in increased VEGF expression have been tested in ALS animal models.
One of these studies delivered AAV4-VEGF and AAV4-insulin-like growth factor (IGF1)
into the lateral and 4th ventricles of SOD1 mice. Administered individually both factors
delayed motor decline and extended survival; however, when delivered in combination the
therapeutic effect was not cumulative. An alternative approach might be to increase
endogenous VEGF production.

Figure 2: Gene Therapy trials for neurodegenerative disorders (Gene therapy for Neurodegenerative

Diseases, Deirdre M. O’Connor)

 xxi

CONCLUSION
Gene therapy has the potential to significantly advance the treatment of neurodegenerative
diseases. However, success in bridging the gap between promising proof-of-principle
concepts to therapeutic efficacy in clinical trials has remained elusive. Initial trials have
demonstrated that delivery of gene therapies to the CNS is safe and well-tolerated. To
improve delivery, new vectors have been identified and developed. An example is AAV9,
which can cross the BBB and has a strong neuronal tropism. Although it must be noted that
the transduction pattern of AAV9 has been shown to change from neurons to astrocytes in
mice depending on their age at administration. In addition, the manufacturing costs to provide
sufficient AAV9 for an intravenous delivery in human trials are significant. A further area
where progress is being made is in the methods of delivery for gene therapies to the CNS.
Delivery methods such as ICV, intrathecal, and direct injection into the brain and spinal cord
are being developed and refined. Improvements in vectors and delivery methods will only
show an effect if the therapeutic gene selected is efficacious. The most significant area of
development is in the identification and testing of new therapeutic genes. This is based on a
better understanding of disease initiation and progression. Better understanding of the
ethology of neurodegenerative diseases will also likely lead to earlier diagnosis, which will
allow intervention before the targeted cells are lost. One area where this is being pursued is in
the identification and validation for bio-markers of neurodegenerative diseases. The
combination of these advances will help to translate new gene therapies to the clinic, yielding
true improvements in treating these devastating diseases.
 xxii

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Neurology, Volume 7, Issue 5, 2008, Pages 400-408
6. Aikaterini Nanou, Mimoun Azzouz, Gene therapy for neurodegenerative
diseases based on lentiviral vectors, Editor(s): Joost Verhaagen, Elly M. Hol,
 xxiii

Inge Huitenga, Jan Wijnholds, Arthur B. Bergen, Gerald J. Boer, Dick F. Swaab,
Progress in Brain Research, Elsevier, Volume 175, 2009, Pages 187-200
7. O’Connor, D. M., & Boulis, N. M. (2015). Gene therapy for neurodegenerative
diseases. Trends in molecular medicine, 21(8), 504-512.
8. Nanou, A., & Azzouz, M. (2009). Gene therapy for neurodegenerative diseases
based on lentiviral vectors. Progress in brain research, 175, 187-200.
9. Sudhakar, V., & Richardson, R. M. (2019). Gene therapy for neurodegenerative
diseases. Neurotherapeutics, 16(1), 166-175.
10. Shihabuddin, L. S., Palmer, T. D., & Gage, F. H. (1999). The search for neural
progenitor cells: prospects for the therapy of neurodegenerative disease.
Molecular medicine today, 5(11), 474-480.
11. Carter, J. E., & Schuchman, E. H. (2001). Gene therapy for neurodegenerative
diseases: fact or fiction? The British Journal of Psychiatry, 178(5), 392-394.
12. Qu, Y., Liu, Y., Noor, A. F., Tran, J., & Li, R. (2019). Characteristics and
advantages of adeno-associated virus vector-mediated gene therapy for
neurodegenerative diseases. Neural regeneration research, 14(6), 931.
13. Lim, S. T., Airavaara, M., & Harvey, B. K. (2010). Viral vectors for neurotrophic
factor delivery: a gene therapy approach for neurodegenerative diseases of
the CNS. Pharmacological Research, 61(1), 14-26.
14. Massaro, G., Mattar, C. N., Wong, A. M., Sirka, E., Buckley, S. M., Herbert, B.
R., ... & Richard-Londt, A. (2018). Fetal gene therapy for neurodegenerative
disease of infants. Nature medicine, 24(9), 1317-1323.
 1

WEEKLY PROGRESS REPORT

(BTB 7th SEMESTER)
For the week: 01 September 2020 – 07 September 2020

WPR No. 1
Name Shreeya Sharma
Enrollment No. A0504117075
Roll no. BTB/17/156
Course: B. Tech Biotechnology
Title of Report Gene Therapy for Neurodegenerative disorders
Faculty Guide Dr. Maansi Vermani

TARGETS FOR THIS WEEK

To various literatures, reports, articles, journals to try and understand this topic to a greater extent

PROGESS OF THE WEEK

1. O’Connor, D. M., & Boulis, N. M. (2015). Gene therapy for neurodegenerative diseases.
2. Nanou, A., & Azzouz, M. (2009). Gene therapy for neurodegenerative diseases based on
lentiviral vectors.
3. Sudhakar, V., & Richardson, R. M. (2019). Gene therapy for neurodegenerative diseases..
4. Shihabuddin, L. S., Palmer, T. D., & Gage, F. H. (1999). The search for neural progenitor cells:
prospects for the therapy of neurodegenerative disease.
 2

WEEKLY PROGRESS REPORT

(BTB 7th SEMESTER)
For the week: 08 September 2020 – 14 September 2020

WPR No. 2
Name Shreeya Sharma
Enrollment No. A0504117075
Roll no. BTB/17/156
Course: B. Tech Biotechnology
Title of Report Gene Therapy for Neurodegenerative disorders
Faculty Guide Dr. Maansi Vermani

PROGESS OF THE WEEK

 Carter, J. E., & Schuchman, E. H. (2001). Gene therapy for neurodegenerative diseases: fact
or fiction?
 Qu, Y., Liu, Y., Noor, A. F., Tran, J., & Li, R. (2019). Characteristics and advantages of adeno-
associated virus vector-mediated gene therapy for neurodegenerative diseases.
 Lim, S. T., Airavaara, M., & Harvey, B. K. (2010). Viral vectors for neurotrophic factor
delivery: a gene therapy approach for neurodegenerative diseases of the CNS.
 Wrote ‘introduction’
 3

WEEKLY PROGRESS REPORT

(BTB 7th SEMESTER)
For the week: 15 September 2020 – 21 September 2020

WPR No. 3
Name Shreeya Sharma
Enrollment No. A0504117075
Roll no. BTB/17/156
Course: B. Tech Biotechnology
Title of Report Gene Therapy for Neurodegenerative Disorders
Faculty Guide Dr. Maansi Vermani

PROGESS OF THE WEEK

 Wrote Literature Review
 Revised Introduction
 Wrote Abstract
 4

WEEKLY PROGRESS REPORT

(BTB 7th SEMESTER)
For the week: 20 September 2020 – 25 September 2020

WPR No. 4
Name Shreeya Sharma
Enrollment No. A0504117075
Roll no. BTB/17/156
Course: B. Tech Biotechnology
Title of Report Gene Therapy for Neurodegenerative disorders
Faculty Guide Dr. Maansi Vermani

PROGESS OF THE WEEK

 Wrote Characteristics of Gene Therapy
 Wrote Delivery Vehicles of Gene therapy
 During, M. J., & Leone, P. (1995). Adeno-associated virus vectors for gene therapy of
neurodegenerative disorders. Clinical neuroscience (New York, N.Y.), 3(5), 292–300.
 Mandel, R. J., Spratt, S. K , progressive 6-hydroxydopamine-induced degeneration
model of Parkinson's disease in rats.
 Jonathan Riley, MD, Thais Federici, PhD, Intraspinal Stem Cell Transplantation in
Amyotrophic Lateral Sclerosis
 Marc S. Weinberg, R. Jude Samulski, Thomas J. McCown, Adeno-associated virus
(AAV) gene therapy for neurological disease
 William J Marks, Jill L Ostrem, Safety and tolerability of intraputaminal delivery of
CERE-120 (adeno-associated virus serotype 2–neurturin) to patients with idiopathic
Parkinson's disease
 Aikaterini Nanou, Mimoun Azzouz, Gene therapy for neurodegenerative diseases
based on lentiviral vectors
 5

WEEKLY PROGRESS REPORT

(BTB 7th SEMESTER)
For the week: 25 September 2020 – 30 September 2020

WPR No. 5
Name Shreeya Sharma
Enrollment No. A0504117075
Roll no. BTB/17/156
Course: B. Tech Biotechnology
Title of Report Gene Therapy for Neurodegenerative disorders
Faculty Guide Dr. Maansi Vermani

PROGESS OF THE WEEK

 Wrote delivery methods
 Wrote fundamental modes of gene therapy
 Recent developments in Parkinson’s disease gene therapy
 Revised report