





































��

Berkeley
Pharma Tech
Journal of Medicine

Correspondence:� 
ICIBHXBU�CQU!HNBJM�DPN

Keywords:
*TDIFNJD�TUSPLF
/FVSPHFOFTJT
(FOF�UIFSBQZ   
/FVSPUSPQIJD�GBDUPST�           
/FVSP%1
/FVSPHFOFSBUJPO�
7FDUPST

Submitted�.BZ��
������ 
Accepted�+VOF���
����� 
1VCMJTIFE�July���
�����

Full Open Access

Creative Commons Attribution� 
License 4.0

Abstract
Stroke is one of the predominant causes of long-term disability and death, and 
is characterized by the cessation of blood flow to part of the brain, preventing it 
from accessing oxygen and nutrients needed for ATP production and proper 
function. Ischemic stroke, the most prevalent type of stroke, involves an obstruction 
such as a blood clot in a blood vessel. Current treatments seek to remove the 
obstruction and restore blood flow, but are time-limited and do not reverse tissue 
damage once it has occurred. The endogenous response to ischemic stroke 
involves an increase in neurogenesis but falls short in producing functional 
recovery. Gene therapy that enhances neurogenesis has provided encouraging results 
in animal models. Preclinical studies in this area have utilized neurotrophic factors 
that can promote migration of cells from the subventricular zone (SVZ). The 
transcription factor NeuroD1 converts reactive glial cells, which increase during 
ischemia, into neurons that integrate into the existing circuits of the brain. The 
clinical value of gene therapy depends upon the development of safe and efficient 
administration methods. In this review, we draw from scientific literature to evaluate 
the relevant genes and vectors for treating ischemic stroke through neurogenesis, and 
discuss strategies to overcome current limitations of gene therapy in human patients.

/FVSPHFOFTJT: Gene-Based Strategies for 
5SFBUJOH�*TDIFNJD�4USPLF
�ïw�¡èßéÞßáÛéÞ��Þ×Ýí×êu�¬åæÞßÛ��èåäéêÛßäu��ä×è��Þ×êêu�×äÚ�¯×äâå×ä�§ÝëïÛä



*OUSPEVDUJPO

Stroke is a leading cause of death, accounting for 5.5 million deaths 
worldwide each year1. In the United States, stroke is attributed to 1 of 
every 19 deaths, and one person will su�er from a stroke every 40 
seconds. While 80% of those who su�er from strokes survive, many are 
impaired with permanent disability. Although several types of strokes 
exist, including ischemic, hemorrhagic and transient ischemic attacks, the 
majority of research is focused on ischemic stroke, which makes up 80% 
of cases2. Ischemic stroke results from an obstruction of blood vessels 
that lead to the brain and causes an impairment of function1. This arises 
from atherosclerosis due to cerebral thrombosis, a blood clot that 
develops at the fatty deposits, or cerebral embolism, a blood clot that 
forms in large arteries in the neck, chest or heart and travels into the 
smaller blood vessels of thebrain3.�Based�on� the�duration� that� the�brain� is� 
deprived� of� blood� and� the� region� that� is� blocked,� strokes� can� bring� 
about� temporary� or�permanent�disabilities, if not death1.

While�stroke�continues�to�be�among�the�most�debilitating�illnesses,�current� 
treatments� for� stroke�are� limited�by� their�time�of�administration�and�most� 
notably� cannot� reverse� tissue� damage� once� it� has� occurred.� The� most� 
prominent�and�vastly�used� ischemic� stroke� treatments�created� thus� far�are� 
emergency� interventions� given� to� stroke� patients� within� a� few� hours� of� 
stroke� onset,� and� are� limited� in� safety� and� availability.� These� include� 
thrombolysis,� the� chemically� induced� breakdown� of� blood� clots,� and� 
thrombectomy,� the� surgical� removal�of� the�blood�clots�with�a�catheter4.�A� 
stroke� patient� must� receive� the� standard� thrombolytic� treatment,� tissue� 
plasminogen�activator�(tPA),�within�4�½�hours�of�onset,�and�patients�above� 
80�years�of�age�must�receive�tPA�within�3�hours5.�Moreover,�tPA�comes�with� 
the�risk�of�hemorrhage,�neurologic�decline�and�orolingual�angioedema6,�and� 
its�positive�outcome� rate� is�estimated�to�be�only�43%5.�Thrombectomy�is�a� 
more�versatile�option�because�of�its�ability�to�treat�patients�with�a�proximal� 
occlusion,� unlike� thrombolysis.� Still,� it� is� restricted� by� a� six-hour� window� 
from� the� onset� of� symptoms� and� is� associated� with� risks� such� as� 
hemorrhage, reocclusion and cerebral edema7,8.

Berkeley Pharma Tech Journal of Medicine | 31



Neurogenesis

A strategy for treating ischemic stroke by gene therapy involves increasing 
neurogenesis, the process by which neurons are formed by neural stem cells 
(NSCs). Neurogenesis is the basis of cognitive function and allows the brain 
to respond to new stimuli9. Through constant re�ning and moderation of 
these neurons, the brain is able to adapt to cognitive, environmental, and 
pathophysiological demands. During prenatal and embryonic stages of 
human development, the majority of neurons are formed, but the process is 
continued through adulthood at a decreased rate in localized zones10.

During prenatal neurogenesis, the central nervous system (CNS) consists of 
NSCs, speci�cally radial glial cells (RGCs), that generate neurons11. The 
RGCs occupy a majority of the prenatal ventricular zone and move from 
symmetric to asymmetric cell division. As asymmetric division and 
proliferation occurs, the RGCs separate, allowing for the creation of neural 
progenitor cells (NPCs) and intermedial neuronal precursors. After further 
separation, new neurons are formed12. As these neurons migrate through 
the embryo, they generate neural circuitry and increase in maturation. The 
rate of this prenatal neurogenesis and the type of neurons formed depend 
on genetic and molecular factors, although prenatal neurogenesis in humans 
typically occurs at gestational week 5 and is thoroughly developed by week 
3413.

Although the majority of neurogenesis occurs before a human is born, the 
neurogenic processes that occur throughout the lifespan continue to play a 
signi�cant role in brain function9. In contrast to prenatal neurogenesis, 
which occurs throughout the entire CNS, neurogenesis during adulthood 
occurs primarily in the subventricular zone (SVZ) and subgranular zone 
(SGZ)15. Stem cells formed during adulthood do not spontaneously 
activate, but instead are dormant in the brain and only activate in response 
to new cognitive or environmental signals16. Along with the lack of 
spontaneous growth and decreased proliferation of neurons during 
adulthood, the density, plasticity, and ability to repair neurons also 
diminishes over time17. These factors all contribute to a decreased ability of

Berkeley Pharma Tech Journal of Medicine | 32



the brain to recover after injury. Because neurological impairments, 
including those caused by ischemic stroke, are more prevalent among older 
populations, the creation of healthy neurons after neurological injury is 
rarely su�cient to induce functional recovery18.

Pathophysiology of Ischemic Stroke
In ischemic stroke, the obstruction of blood vessels to the brain limits 
cerebral oxygen and glucose supply, causing tissue damage. This reduction 
in oxygen and glucose due to inadequate cerebral blood �ow initiates 
biochemical cascades that lead to cell death19. Oxygen and glucose are 
required to produce ATP, the key source of energy in cells, and as a result of 
ischemia, ATP levels decline20. This prevents the function of ion pumps 
that rely upon ATP to maintain ionic gradients. Failure of the Na+/K+ 

ATPase pump, which normally transports K+ into the cell and Na+ out of 
the cell, causes excess intracellular Na+ and extracellular K+. Intracellular 
Ca2+ levels rise because of Ca2+ pump failure21, and the binding of Ca2+ to 
synaptotagmin stimulates glutamate release to the synapse. Excess glutamate 
leads to the hyperexcitation of NMDA glutamate receptors, providing 
positive feedback for intracellular Ca2+ levels22. In a phenomenon known as 
excitotoxicity, excess glutamate sets the stage for mechanisms of cell death. 
Excess intracellular Ca2+ levels activate endonucleases, lipases, phosphatases 
and proteases23, which contribute to cell membrane disruption, cytoskeletal 
breakdown, DNA fragmentation, free radical production and 
mitochondrial damage. These processes lead to cell death by apoptosis or 
necrosis and irreversible tissue damage19.

Berkeley Pharma Tech Journal of Medicine | 33



Figure 1: Pathway from ischemic stroke to tissue damage in the
brain. Obstruction of blood vessels leading to the brain reduces oxygen and
glucose supply, which are required for ATP production. Ca2+ and Na+/K+

pumps rely on ATP and their failure in ischemic conditions causes ion
imbalances. Excess intracellular Ca2+ leads to glutamate excitotoxicity and cell
death.

Ischemic stroke additionally results in neuroin�ammation. Inadequate
blood supply activates microglia, leading to in�ammation through cytokine
upregulation, disruption of the blood-brain barrier (BBB) and the
production of reactive oxygen species. In astrocytes, in�ammatory factors
like glial �brillary acidic protein (GFAP) result in reactive gliosis and glial
scar formation, and failure of the Na+/K+ ATPase pump leads to the
swelling of astrocytes. Ischemia obstructs the connection between astrocytes
and endothelial cells at the BBB, allowing peripheral in�ammatory cells to
breach24.

The glial scar—primarily made up of astrocytes and microglia—forms to
protect the brain after stroke but prevents the regeneration of neurons. In

Berkeley Pharma Tech Journal of Medicine | 34



ischemic stroke, astrocytes undergo morphological and functional changes,
proliferating and overlapping with each other during reactive gliosis while
GFAP is upregulated. The scar has neuroprotective e�ects, such as
providing a barrier between healthy and damaged tissue and limiting the
spread of tissue damage25,26. However, it impedes the restoration of neural
function in stroke25 because axons physically cannot cross the scar, and
reactive astrocytes produce growth-inhibiting factors that inhibit axon
extension. While eliminating reactive astrocytes increases tissue damage, due
to their bene�ts15, strategies to mitigate the obstructive e�ects of the scar
and restore long-term function would be favorable in treating stroke.

Neurogenesis increases as an endogenous response to ischemic stroke, but
this is insu�cient to restore function after stroke27. This process is driven by
NSCs in the SVZ, which proliferate and di�erentiate due to an
upregulation of neurotrophic factors28. The mechanism behind NSC
proliferation and di�erentiation in post-stroke neurogenesis is reported to
be induced by H19, a long non-coding RNA29. Stroke patients experience a
level of functional improvement in the �rst few months following stroke
due to self-repair30, so enhancing neurogenesis would be a promising
approach to improve function after stroke.

In vivo animal models are typically used for preclinical research of ischemic
stroke and allow researchers to replicate the pathophysiology of the
condition in humans with far more complexity than in vitro models31. The
standard method to model focal ischemic stroke in animals is through the
occlusion of the middle cerebral artery (MCA)—which is easily accessible
and the most common site of stroke in humans—by mechanical devices,
blood clots or pharmacological agents32,33. MCA occlusion is often induced
by inserting an intraluminal thread into the internal carotid artery via the
external carotid artery to block the MCA. This leads to signi�cant damage
in local brain tissue within three hours but can be lacking in precision,
despite its non-invasiveness. More direct access to the MCA requires
craniectomy, the surgical opening of the skull, which exposes the brain to
the atmosphere and poses a risk for cerebrospinal �uid leakage, infection
and unintended damage34.

Berkeley Pharma Tech Journal of Medicine | 35



Gene Therapy to Enhance Neurogenesis
To induce neurogenesis by gene therapy, genes are typically administered 
through a vector. To successfully deliver a gene in vivo, we would have to 
identify the appropriate transgenes for the local recruitment of endogenous 
stem cells. This technique could be used to slow the spread of neural decline 
or generate a neural niche in regions that did not already express the 
necessary signals or factors35. Vectors are preferred over the delivery of naked 
DNA because of their greater e�ciency, and viral vectors take advantage of 
viruses’ evolved ability to alter gene expression in cells they infect36. Viruses 
deliver genetic information by infecting the cell directly, but in gene therapy, 
they are modi�ed so they are unable to transfer unwanted information and 
result in pathogenicity. Delivery of vectors is carried out in two ways: in 
vivo, where the gene is delivered directly into the patient, and ex vivo, where 
cells are removed from the patient before gene delivery and then inserted 
back into the patient37.

Figure 2: Schematic of gene therapy methods. In vivo gene therapy often
involves direct delivery of a vector into the body to alter gene expression. Ex vivo
gene therapy is performed by removing cells from the body, delivering the
vector into cells and transplanting altered cells into the body.

Berkeley Pharma Tech Journal of Medicine | 36



Each viral vector o�ers unique advantages in gene therapy but has particular
drawbacks. A class of vectors that is rising in popularity in gene therapy
utilizes the adeno-associated virus (AAV)38 because of its low
immunogenicity and thus its propensity for an undesired immune response.
Integration of AAV into the host genome depends on the presence of the
rep gene, which exists in wild-type AAV. Recombinant AAV (rAAV) does
not contain rep and therefore helps limit side e�ects39. A key disadvantage
of AAVs that do not integrate into the host genome is that subsequent
doses, if needed, would have a reduced e�cacy due to an adaptive immune
response. To combat this, methods to remove anti-AAV antibodies from the
bloodstream, including plasmapheresis and IgG-cleaving endopeptidases,
have been developed and shown e�ectiveness37. Serotypes of AAV have also
been engineered to improve its permeability to the BBB, which is necessary
for peripheral administration in treating CNS disorders like stroke40. For
example, AAV-PHP.eB has the ability to transduce most neurons in the
cortex and striatum and e�ciently promote gene expression in the CNS41.

Another viral vector being investigated for use in ischemic stroke models is
the adenoviral vector, which o�ers greater capacity and transfection
e�ciency. A study of adenovirus-mediated gene transfer to an ischemic
brain model found that this vector provided e�ective expression of
transgene at the nonischemic and peri-ischemic areas42. However, adenoviral
vectors are limited by their high immunogenicity, and high doses can lead to
in�ammation43. Lentiviral vectors, derived from the retrovirus family, have
lower immunogenicity than other retrovirus-based vectors and most
notably integrate into the host genome. This ability makes them useful for
stimulating stable long-term expression. At the same time, genomic
integration puts target cells at risk for insertional mutagenesis44. Unlike
lentiviral vectors, retroviral vectors have limited use in gene therapy to
promote neurogenesis because they only transduce dividing cells, and
neurons are non-dividing cells45. All viral vectors have advantages and
drawbacks and should be chosen on a case-by-case basis, but rAAV would
generally be favorable to enhance neurogenesis in humans. Low
immunogenicity should be prioritized from a therapeutic standpoint, and
this is a de�ning characteristic of rAAV.

Berkeley Pharma Tech Journal of Medicine | 37



Although less common than viral vectors, another area of interest in gene 
therapy for stroke is the delivery of genes by non-viral means. In general, 
non-viral vectors limit the risk of cytotoxicity, immunogenicity and 
mutagenesis, making them an attractive alternative to viral vectors for 
therapeutic use in humans, while being less costly. Their e�ectiveness is 
hindered by their relatively low transfection e�ciency, speci�city and 
duration of expression46. The conventional method of non-viral gene 
delivery is polyethylenimine (PEI), a polymer that is associated with 
cytotoxic risks due to its high positive charge density47. Conjugation of PEI 
with deoxycholic acid to carry the anti-in�ammatory gene heme-oxygenase 1 
can help to overcome this challenge and reduce infarct size from ischemic 
stroke48. Despite the potential of non-viral vectors, their viral counterparts 
remain the most prevalent type of vector in research and existing drugs 
because of their e�ciency, diversity and adaptability49.

Neurotrophic Factors in Gene Therapy
Neurotrophic factors promote the growth and survival of neurons and are 
the primary group of molecules that can be overexpressed through gene 
therapy to induce neurogenesis. They serve an array of other functions in 
treating stroke50. Neurotrophic factors play roles in the development of 
axons, dendrites and synapses51, and are endogenously upregulated in 
response to stroke52. Neurotrophins, a family of neurotrophic factors, target 
transmembrane receptors Trk and p75-NTR. Neurotrophins bind, causing 
dimerization and transphosphorylation of Trk. This activates PLCγ, which 
produces IP3 and DAG to enhance intracellular Ca2+ release and promote 
synaptic plasticity via protein kinase C. The growth of axons and dendrites 
is mediated by a separate pathway featuring Trk, Ras and ERK. The PI3-K 
pathway activates serine/threonine kinase to drive neuronal survival53. 
Several neurotrophic factors that have been investigated in gene therapy 
research pertaining to ischemic stroke, and their bene�ts and limitations, 
will be assessed in this section.

Berkeley Pharma Tech Journal of Medicine | 38



The �rst neurotrophic factor to be discovered and the one that is most
well-understood is nerve growth factor (NGF)50, a neurotrophin that
promotes both neurogenesis via upregulation of growth-associated protein
43 (GAP-43) and angiogenesis via upregulation of vascular endothelial
growth factor (VEGF)54. Serum NGF levels are inversely correlated with
post-stroke function, potentially indicating a therapeutic link55.
Pseudolentiviral gene therapy with β-NGF, a subunit of the factor with very
similar e�ects, in the rat hippocampus increases neurogenesis and reduces
apoptosis following ischemic stroke, as reported by Cao et al. (2018).
Cognitive functional recovery was demonstrated by improved Morris water
maze performance of rats that received β‐NGF56. Non-invasive routes of
NGF administration have been investigated as well. In a study by Zhu et al.
(2011), intranasal delivery in rats improved survival in the striatum and SVZ
but failed to enhance cell proliferation in those regions57. NGF has been
explored in relation to non-viral vectors, in part to overcome the
impermeability of NGF to the blood-brain barrier. In vitro carbon
nanotube delivery showed that NGF treatment promotes neuron survival in
a sustained manner, although its in vivo applications remain unclear58.
Albumin nanocarriers successfully promoted neurite growth in vitro.
However, in vivo delivery of NGF in combination with U0126, which
inhibits the MEK pathway involved in ischemia, did not signi�cantly reduce
infarct size compared to U0126 alone, suggesting that this treatment is
lacking in e�cacy59. NGF delivery with exosomes in the ischemic cortex of
mice resulted in long-lasting neuroprotection and indications of
neurogenesis, but functional recovery was not tested60. Clinical trials of
NGF in Alzheimer’s disease and Parkinson’s disease patients revealed
negative side e�ects, such as weight loss and back pain due to nociceptive
activation by NGF. The e�ect of NGF on nociception must be mitigated
and methods for e�cient transport across the BBB must be developed to
make it an e�ective therapeutic option61.

Brain-derived neurotrophic factor (BDNF), the most abundant
neurotrophin, is another factor that induces neurogenesis and helps prevent
apoptosis. Overexpression of BDNF protects neurons from glutamate
excitotoxicity and has been looked at as a potential therapeutic option in
stroke patients62. At the same time, excess BDNF in the forebrain can have

Berkeley Pharma Tech Journal of Medicine | 39



negative e�ects on learning and memory63. As with NGF, stroke is
associated with a reduction in BDNF levels in the bloodstream64.
AAV-BDNF gene therapy targeting the SVZ, a key location of adult
neurogenesis, of rats two weeks before contralateral MCA occlusion
increases the migration of NPCs from the SVZ and contributes to
behavioral recovery. It failed to reduce infarct size signi�cantly, contradicting
the �ndings of previous studies into the factor65. Gene therapy utilizing
BDNF has also been used to reduce symptoms of stroke in preclinical
research, including post-stroke pain and depression. This suggests that
BDNF plays roles in both enhancing neurogenesis and reducing
in�ammation66,67. A clinical trial conducted in 1999 tested the e�ectiveness
of BDNF, administered subcutaneously, in patients with amyotrophic
lateral sclerosis, a neurodegenerative disease. Although side e�ects were
minimal, the trial was unable to establish a signi�cant bene�t of BDNF
treatment. Still, it had success in patients with early respiratory impairment
and altered bowel function, a side e�ect of the treatment, indicating a need
for further research68. Future trials with BDNF would likely bene�t from
more re�ned methods of gene transfer into subjects.

Neurotrophin-3 (NT-3) is part of the same family as NGF and BDNF and
plays a distinct role in neurogenesis during development53. It is involved in
the survival, proliferation and di�erentiation of neurons, as well as in the
plasticity and regeneration of glutamatergic neurons. NT-3 is a contributor
to revascularization and has anti-in�ammatory properties69. Duricki et al.
(2016) investigated the e�ectiveness of NT-3 in gene therapy treatment for
stroke. AAV-mediated intramuscular delivery of the factor in adult and
elderly rats 24 hours after stroke resulted in neuroplasticity, as observed
through the sprouting of corticospinal axons, and behavioral recovery.
Neuroprotection was not displayed due to the delay in treatment, and rats
experienced minor in�ammation from AAV injection70. NT-3 binds to
TrkC receptors, which are not found on adult nociceptors, making it
advantageous over the pain-regulating NGF. Duricki et al. (2019) con�rmed
this bene�t in the forepaws of adult and elderly rats. Furthermore, human
clinical trials in patients with neuropathy have not revealed signi�cant side
e�ects71. NT-3 appears to be a safer alternative to other members of the

Berkeley Pharma Tech Journal of Medicine | 40



neurotrophin family, and future research into its e�ect on neuron survival
could reinforce a need for clinical studies in stroke patients.

Neurotrophic factors that are not part of the neurotrophin family have also
attracted the attention of researchers in gene therapy. Among these is ciliary
neurotrophic factor (CNTF), an important contributor to endogenous
neurogenesis that occurs after stroke. CNTF promotes proliferation of cells
from the SVZ72. Its continuous administration into the lateral ventricle for
four weeks after MCA occlusion in rats had the capacity to reduce infarct
size of the cortex and degeneration of the thalamus, while improving spatial
learning73. MacLaren et al. (2006) found that CNTF has protective e�ects
on ganglion cells, following intravitreal administration of AAV-CNTF74.
Although not directly related to ischemic stroke, this study reveals the
therapeutic applications of CNTF in preventing tissue damage from CNS
injury that occurs in ischemic stroke. A clinical trial by Chew et al. (2019)
using CNTF in patients with macular telangiectasia, a neurodegenerative
condition of the retina, had success in promoting retinal neuroprotection75.
While this trial showed minimal side e�ects, it is important to note in future
human applications that CNTF and its neurogenic e�ects are linked to
satiety and weight loss76.

Glial cell-derived neurotrophic factor (GDNF) promotes neurogenesis in
striatal neurons and is involved in synaptic plasticity. It primarily acts on
dopaminergic neurons, stimulating the release of the neurotransmitter
dopamine in the striatum77. Dopamine plays a major role in sensorimotor
function, and research shows that ischemic stroke contributes to motor
de�cits through the loss of dopaminergic neurons78. Beker et al. (2022)
reported that intracerebral lentiviral delivery of GDNF 10 days before
MCA occlusion led to an increase in the expression of the dopaminergic
neuron transcription factor Nurr1 and reduced degeneration of neurons in
mice. The treatment did not appear to increase the number of striatal
dopaminergic neurons following ischemic stroke77. Beker et al. (2020)
observed that lentiviral GDNF delivery in mice induces both neurogenesis
and angiogenesis and reduces the glial scar in the peri-infarct region. In
addition, motor recovery was demonstrated79. Clinical trials using GDNF to

Berkeley Pharma Tech Journal of Medicine | 41



treat Parkinson’s disease have not shown a signi�cant improvement in
motor function compared to a placebo group. Trials in which GDNF was
delivered directly to the putamen had greater success, indicating that it
could be useful through more e�cient routes of administration80.

Insulin-like growth factor 1 (IGF-1) contributes to neurogenesis and
angiogenesis. Low levels of IGF-1 are associated with more severe tissue
death in stroke patients, and it is produced as part of the endogenous
response to stroke. Zhu et al. (2009) intracerebrally delivered AAV-IGF-1
after MCA occlusion, which was shown to promote neurogenesis by
enhancing proliferation and migration of NSCs from the SVZ. Its ability to
increase vascular density promotes cerebral blood �ow81. Okoreeh et al.
(2017) reported that AAV5-IGF-1 six to eight weeks before MCA occlusion
was unable to reduce infarct size by targeting astrocytes. Behavioral recovery
and a reduction in neuroin�ammation were still observed82. The e�ect of
IGF-1 on neurogenesis to treat diseases like stroke is yet to be clinically
researched. Future preclinical studies are required to investigate its
neuroprotective abilities before application in humans.

Vascular endothelial growth factor (VEGF) primarily induces angiogenesis
but is also known to enhance neurogenesis in the SVZ and SGZ. Wang et al.
(2007) shed light on the connection between VEGF overexpression and
neurogenesis. Liposomes, a non-viral vector, were used to transfer VEGF
into the lateral ventricle of the ischemic rat brain, increasing striatal
neurogenesis and reducing the size of infarction83. Markosyan et al. (2020)
tested adenoviral vectors carrying VEGF in combination with GDNF and
neural cell adhesion molecule as a preventative option for ischemic stroke in
rats. Intrathecal delivery reduced infarct size and brought about functional
recovery of neurons, though neurogenesis was not observed84. Acute
upregulation of VEGF is associated with disruption of the BBB, which
must be considered for clinical trials and could be mitigated by combining
VEGF with other angiogenic factors85.

Berkeley Pharma Tech Journal of Medicine | 42



Conversion of Glial Cells to Neurons
A related approach to promote the formation of neurons after stroke 
involves neuroregeneration through the conversion of reactive glial cells, 
which proliferate in the post-stroke brain, into neurons. The endogenous 
response to ischemic stroke features the formation of the glial scar, largely 
made up of reactive astrocytes and microglia, which is driven by GFAP25,26. 
Considering the hindering e�ects of the scar and the low auto-regenerative 
abilities of the CNS, it would be favorable to convert components of the 
scar into neurons. Inducing neurogenesis through the conversion of reactive 
glial cells would both reduce the size of the glial scar and potentially restore 
neuronal function. The neural transcription factor neurogenic 
di�erentiation 1 (NeuroD1), which plays a major role in prenatal 
neurogenesis, has been the primary focus of gene therapy research aiming to 
convert glial cells into functional neurons as a treatment for stroke86. 
NeuroD1 binds to regulatory elements of neuronal development genes to 
promote neuronal migration and transcription via conversion of 
heterochromatin to euchromatin, a process that resembles the maturation 
of neurons seen in the prenatal stage86,87. Although several other 
transcription factors have been identi�ed, including Neurogenin-2, Olig2 
and Ascl1, and have been experimented in di�erent combinations, they do 
not display as much e�ciency or e�cacy as NeuroD1 alone88.

In vivo NeuroD1-based gene therapy studies have shown that the
conversion of reactive glial cells to neurons promotes functional recovery
from ischemic stroke. A study by Ge et al. (2019) using non-human
primates (NHPs), which model the human brain more closely than rodents,
showed that the overexpression of NeuroD1 in reactive astrocytes of the
cortex successfully led to their conversion into neurons. After stroke
induction, it was revealed that NeuroD1-AAV increased neuronal density
and synaptic and dendritic markers, while protecting parvalbumin
interneurons, in treated areas. A 90% reduction in targeted astrocytes was
observed, as well as decreases in reactive microglia and macrophages. These
changes were initiated between 10 and 30 days after the onset of stroke and
lasted between two months and one year, suggesting that
NeuroD1-mediated gene therapy is long-lasting and has a more generous

Berkeley Pharma Tech Journal of Medicine | 43



time window than existing treatments89. Jiang et al. (2021) reported that
lentiviral delivery of NeuroD1 into the peri-infarct region of mice, one week
following stroke onset, resulted in 66% of infected cells being marked as
mature neurons, reduction in astrogliosis, and increases in synaptic
plasticity and sensorimotor function88. A study of NeuroD1-AAV in mice
by Chen et al. (2020) showed the ability of this factor to promote functional
recovery from ischemic stroke, with 30% to 40% of neurons in the motor
cortex being regenerated after NeuroD1 was expressed in reactive
astrocytes90. Altogether, preclinical research into NeuroD1 has been
encouraging, because it accomplishes two critical objectives in reversing
tissue damage from stroke: reducing the obstructiveness of the glial scar and
increasing neurogenesis. Nonetheless, gene therapy with NeuroD1 must be
performed in a regulated manner, because eliminating the glial scar leads to
adverse outcomes15.

Practical Considerations
While preclinical in vivo studies have shown success in promoting 
functional recovery from ischemic stroke through gene therapy, several 
limitations still exist that prevent moving forward with treatments in 
humans. There have been few clinical trials in humans, many of which have 
produced inconclusive or discouraging results. Although studies involving 
rodents and NHPs serve as an important basis for our understanding of 
gene therapy, the di�erences between their genomes from humans could 
create unsuccessful results in the clinical stage.

One of the main obstacles in using gene therapy to treat CNS disorders like 
stroke in humans is the BBB. The vast majority of drugs including the 
factors discussed cannot pass through this barrier, but to minimize the 
invasiveness of gene therapy, vectors expressing therapeutic genes must be 
able to cross in an e�cient manner. Therapeutic options that bypass the 
BBB require direct injection into the brain or high peripheral doses and 
present a signi�cant risk to the patient40,91. High doses of AAV to treat 
X-linked tubular myopathy led to liver dysfunction, sepsis and the deaths of 
two patients in a 2020 clinical trial92. Intravenous administration is a 
non-invasive route, though it requires genes to cross the BBB. It additionally

Berkeley Pharma Tech Journal of Medicine | 44



lacks speci�city, increasing the likelihood of o�-target e�ects on peripheral
organs93. The development of vectors that can e�ciently cross the BBB
would improve the feasibility of intravenous gene therapy. Research in this
area has largely focused on serotypes of AAV, such as AAV-PHP.eB,
although these variants reduce the already small packaging capacity of the
vector91,93. In more recent strategies, vectors cross the BBB by paracellular
transport through disruption of the barrier or by attaching to a transport
receptor94. Intranasal delivery has received interest as a minimally invasive
method of bypassing the BBB95. NGF has been administered intranasally in
rat stroke models and had success in promoting neurogenesis54.

Another aspect that must be considered is the risk for viral-mediated gene
therapy treatment to provoke an immune response. Gene therapy with
rAAV is promising for in vivo treatment of CNS diseases such as stroke
because it is associated with low immunogenicity and no integration into
the host genome39. However, this lack of integration is also a key drawback
of rAAV, because anti-AAV antibodies are produced by the adaptive
immune system, attenuating subsequent doses37. Existing approaches to
remove these antibodies from the bloodstream, as outlined earlier, would
make rAAV gene therapy far less practical in humans. Inducing
neurogenesis through gene therapy may additionally target unwanted cells,
allowing for potentially harmful results. Vectors that can integrate into the
host genome, such as lentiviral and wild-type AAV vectors, present the
greatest risk for insertional mutagenesis44. Therefore, the use of vectors that
do not integrate may presently be the safest option.

Safety of gene therapy in humans requires that side e�ects are minimized.
The target cells of genes must be as restricted as possible, which is
dependent on how well the promoters used are able to target desired cells.
Cell-type speci�city of promoters remains a challenge, especially for
intravenous administration93. Gene therapy that enhances neurogenesis to
treat stroke aims to target neurons in the hippocampus and lateral
ventricles, the two primary locations of adult neurogenesis. Finneran et al.
(2021) reported that the promoters CAMKIIα and human synapsin 1 allow
high-level gene expression speci�c to neurons, including those in the

Berkeley Pharma Tech Journal of Medicine | 45



hippocampus, and prevent peripheral expression when used with AAV. Use 
of CAMKIIα resulted in lower aberrant expression in the heart, lung and 
muscle compared to human synapsin 196. For gene therapy utilizing 
NeuroD1, it would be bene�cial to target reactive glial cells to optimize 
their conversion into neurons. The standard promoter used for expression 
in astrocytes, the main component of the glial scar, is GFAP, but its large 
size is unfavorable for AAV, which has a small capacity. gfaABC1D is a 
smaller promoter that also targets astrocytes but research into its speci�city 
has been inconclusive97. Taschenberger et al. (2017) investigated the 
MicroRNA124 (miR124) target sequence, which is speci�c to neurons, as a 
means of reducing o�-target e�ects. The study demonstrated that miR124 
greatly reduces o�-target gene expression in neurons, although the level of 
expression was less than desired98.

Future Directions
Although the factors examined in this review have generally shown promise 
in gene therapy for stroke in the preclinical stage, minimal clinical research 
has been conducted in relation to stroke speci�cally, and success in humans 
has been limited. Key areas of future research that would help open the door 
for gene therapy in humans include vectors that can e�ciently cross the 
BBB and minimize aberrant gene expression. Improving the transfection 
e�ciency of non-viral vectors could potentially make them a more practical 
option than viral vectors for gene therapy in humans, and thus should be an 
area of future research interest. Non-viral vectors are far lower than viral 
vectors in immunogenicity, cytotoxicity and mutagenesis. To make them a 
more practical option in gene therapy to treat stroke, strategies to improve 
their speci�city and transfection e�ciency must be further researched46.

To gain a fuller understanding of ischemic stroke and the e�ectiveness of 
gene therapy as a treatment option, animal stroke models must be re�ned. 
The standard model for focal cerebral ischemia, intraluminal MCA 
occlusion, can lead to hyperthermia by blocking hypothalamic blood supply 
and subarachnoid hemorrhage if the suture is not inserted properly99. Laser 
doppler �owmetry has attracted attention as a way to accurately guide the

Berkeley Pharma Tech Journal of Medicine | 46



suture, but studies into its bene�ts for modeling MCA occlusion have had
mixed results100,101. Alternative models must also be investigated so that new
treatments encompass less common forms of ischemic stroke. Only 50.8%
of ischemic stroke arise from the MCA but the vast majority of in vivo
models used to evaluate gene therapy use MCA occlusion. Small-vessel and
brainstem strokes, for example, combine to make up 24.2% of cases.
Developing techniques to access deeper regions of the brain would help
overcome this disproportion102. Further research into stroke models would
improve the likelihood of translating preclinical successes in gene therapy
into viable treatments for humans.

Gene therapy using neurotrophic factors and NeuroD1 must be carefully
controlled in its duration to avoid the development of tumors.
Accumulating evidence has implicated them in tumor neurogenesis, the
formation and proliferation of cancer cells. NGF is overexpressed in breast,
gastric, liver, lung, ovarian, pancreatic, skin and thyroid cancers. Cancer cells
secrete NGF and contribute to tumor neurogenesis by acting on Trk
receptors on nerves. Moreover, NGF is linked to heightened pain
experienced by cancer patients103. Other neurotrophins, including BDNF,
are upregulated in various types of cancer104. Studies have indicated that
NeuroD1 is expressed in small cell lung and colorectal cancers105,106.
However, the risk of cancer from these factors in gene therapy for stroke is
yet to be thoroughly investigated.

Finally, the enhancement of neurogenesis through gene therapy must be
further investigated in relation to symptomatology. Preclinical studies have
focused on histological markers like growth of axons and dendrites and
proliferation of neurons. Although previous research has indicated that
adult neurogenesis from the hippocampus contributes to learning and
memory107, the direct e�ects of neurogenesis on cognitive recovery from
stroke must be studied in greater detail, and the implementation of new
measures would be bene�cial. The factors discussed in this review had a
wide range of e�ects in reducing the symptoms of ischemic stroke, so the
direct role of neurogenesis remains largely unclear.

Berkeley Pharma Tech Journal of Medicine | 48



Conclusion
As one of the most common causes of death and disability around the 
world, ischemic stroke has become the focus of a growing number of studies 
related to gene therapy. Where current treatments may fail, gene therapy can 
reverse the pathophysiology of stroke and greatly extend the period in which 
patients can be treated after stroke and still experience functional recovery. 
Treatment can be administered to increase neuron proliferation and growth. 
Vectors expressing genes for neurogenic molecules have shown the capacity 
to augment the endogenous response to stroke, which involves low-level 
neurogenesis, in preclinical studies. Overexpression of neurotrophic factors, 
including the neurotrophins NGF, BDNF, NT-3, as well as CNTF, GDNF, 
IGF-1 and VEGF, may have the potential to enhance neurogenesis and aid 
in recovery. However, clinical trials have uncovered side e�ects that should 
be explored in greater detail. From a safety perspective, rAAV is the most 
favorable viral vector for stroke gene therapy due to its low immunogenicity, 
and methods for cell-type speci�city should be re�ned. An alternative 
approach to neurogenesis is the conversion of glial cells to neurons. This 
tactic would employ NeuroD1 to reduce the size of the glial scar while 
simultaneously working to restore neuronal function. To progress further 
from preclinical in vivo studies to treatment in humans, further research is 
required to investigate less invasive methods for e�cient gene therapy. A 
formidable obstacle is the BBB, which is extremely di�cult to cross with 
minimal invasiveness. More research is needed into routes of administration 
and side e�ects to ensure success in human clinical trials.

Berkeley Pharma Tech Journal of Medicine | 49



References

1. Donkor ES. Stroke in the 21st Century: A
Snapshot of the Burden, Epidemiology, and
Quality of Life. Stroke Res Treat.
2018;2018:3238165. Published 2018 Nov 27.
doi:10.1155/2018/3238165

2. Boehme AK, Esenwa C, Elkind MS. Stroke
Risk Factors, Genetics, and Prevention. Circ
Res. 2017;120(3):472-495.
doi:10.1161/CIRCRESAHA.116.308398

3. Chugh C. Acute Ischemic Stroke:
Management Approach. Indian J Crit Care
Med. 2019;23(Suppl 2):S140-S146.
doi:10.5005/jp-journals-10071-23192

4. Gravanis I, Tsirka SE. Tissue-type
plasminogen activator as a therapeutic target in
stroke. Expert Opin Ther Targets.
2008;12(2):159-170.
doi:10.1517/14728222.12.2.159

5. Boehme AK, Esenwa C, Elkind MS. Stroke
Risk Factors, Genetics, and Prevention. Circ
Res. 2017;120(3):472-495.
doi:10.1161/CIRCRESAHA.116.308398

6. Rabinstein AA. Update on Treatment of
Acute Ischemic Stroke. Continuum (Minneap
Minn). 2020;26(2):268-286.
doi:10.1212/CON.0000000000000840

7. Derex L, Cho TH. Mechanical
thrombectomy in acute ischemic stroke. Rev
Neurol (Paris). 2017;173(3):106-113.
doi:10.1016/j.neurol.2016.06.008

8. Krishnan R, Mays W, Elijovich L.
Complications of Mechanical Thrombectomy
in Acute Ischemic Stroke. Neurology.
2021;97(20 Suppl 2):S115-S125.
doi:10.1212/WNL.0000000000012803

9. Ming GL, Song H. Adult neurogenesis in
the mammalian brain: signi�cant answers and
signi�cant questions. Neuron.
2011;70(4):687-702.
doi:10.1016/j.neuron.2011.05.001

10. Mira H, Morante J. Neurogenesis from
embryo to adult – lessons from �ies and mice.
Frontiers in Cell and Developmental Biology.
2020;8. doi:10.3389/fcell.2020.00533

11. Iacopetti P, Michelini M, Stuckmann I,
Oback B, Aaku-Saraste E, Huttner WB.
Expression of the antiproliferative gene TIS21
at the onset of neurogenesis identi�es single
neuroepithelial cells that switch from
proliferative to neuron-generating division.
Proc Natl Acad Sci U S A.
1999;96(8):4639-4644.
doi:10.1073/pnas.96.8.4639

12. Sidman RL, Rakic P. Neuronal migration,
with special reference to developing human
brain: a review. Brain Res. 1973;62(1):1-35.
doi:10.1016/0006-8993(73)90617-3

13. Budday S, Steinmann P, Kuhl E. Physical
Biology of Human Brain Development.
Frontiers in Cellular Neuroscience. 2015;9.
doi:10.3389/fncel.2015.00257

14. Lois C, García-Verdugo JM, Alvarez-Buylla
A. Chain migration of neuronal precursors.
Science. 1996;271(5251):978-981.
doi:10.1126/science.271.5251.978

15. Huang L, Wu ZB, Zhuge Q, et al. Glial scar
formation occurs in the human brain after
ischemic stroke. Int J Med Sci.
2014;11(4):344-348. Published 2014 Feb 11.
doi:10.7150/ijms.8140

16. Lim DA, Alvarez-Buylla A. The Adult
Ventricular-Subventricular Zone (V-SVZ) and
Olfactory Bulb (OB) Neurogenesis. Cold

Berkeley Pharma Tech Journal of Medicine | 50



Spring Harb Perspect Biol. 2016;8(5):a018820.
Published 2016 May 2.
doi:10.1101/cshperspect.a018820

17. Apple DM, Kokovay E. Vascular niche
contribution to age-associated neural stem cell
dysfunction. American Journal of
Physiology-Heart and Circulatory Physiology.
2017;313(5). doi:10.1152/ajpheart.00154.2017

18. Cuartero MI, García-Culebras A,
Torres-López C, et al. Post-stroke
Neurogenesis: Friend or Foe?. Front Cell Dev
Biol. 2021;9:657846. Published 2021 Mar 23.
doi:10.3389/fcell.2021.657846

19. Rama R, García JC. Excitotoxicity and
oxidative stress in acute stroke. Ischemic Stroke -
Updates. October 2016. doi:10.5772/64991

20. Orellana-Urzúa S, Rojas I, Líbano L,
Rodrigo R. Pathophysiology of Ischemic
Stroke: Role of Oxidative Stress. Curr Pharm
Des. 2020;26(34):4246-4260.
doi:10.2174/1381612826666200708133912

21. Khoshnam SE, Winlow W, Farzaneh M,
Farbood Y, Moghaddam HF. Pathogenic
mechanisms following ischemic stroke. Neurol
Sci. 2017;38(7):1167-1186.
doi:10.1007/s10072-017-2938-1

22. Sekerdag E, Solaroglu I, Gursoy-Ozdemir Y.
Cell Death Mechanisms in Stroke and Novel
Molecular and Cellular Treatment Options.
Curr Neuropharmacol. 2018;16(9):1396-1415.
doi:10.2174/1570159X16666180302115544

23. Belov Kirdajova D, Kriska J, Tureckova J,
Anderova M. Ischemia-Triggered Glutamate
Excitotoxicity From the Perspective of Glial
Cells. Front Cell Neurosci. 2020;14:51.
Published 2020 Mar 19.
doi:10.3389/fncel.2020.00051

24. Jayaraj RL, Azimullah S, Beiram R, Jalal FY,
Rosenberg GA. Neuroin�ammation: friend
and foe for ischemic stroke. J
Neuroinflammation. 2019;16(1):142.
Published 2019 Jul 10.
doi:10.1186/s12974-019-1516-2

25. Wang H, Song G, Chuang H, et al. Portrait
of glial scar in neurological diseases. Int J
Immunopathol Pharmacol.
2018;31:2058738418801406.
doi:10.1177/2058738418801406

26. Linnerbauer M, Rothhammer V. Protective
Functions of Reactive Astrocytes Following
Central Nervous System Insult. Front
Immunol. 2020;11:573256. Published 2020
Sep 30. doi:10.3389/�mmu.2020.573256

27. Rahman AA, Amruta N, Pinteaux E, Bix
GJ. Neurogenesis After Stroke: A Therapeutic
Perspective. Transl Stroke Res. 2021;12(1):1-14.
doi:10.1007/s12975-020-00841-w

28. Marques BL, Carvalho GA, Freitas EMM,
et al. The role of neurogenesis in neurorepair
after ischemic stroke. Semin Cell Dev Biol.
2019;95:98-110.

29. Fan B, Pan W, Wang X, et al. Long
noncoding RNA mediates stroke-induced
neurogenesis. Stem Cells. 2020;38(8):973-985.
doi:10.1002/stem.3189

30. Cuartero MI, García-Culebras A,
Torres-López C, et al. Post-stroke
Neurogenesis: Friend or Foe?. Front Cell Dev
Biol. 2021;9:657846. Published 2021 Mar 23.
doi:10.3389/fcell.2021.657846

31. Llovera G, Pinkham K, Liesz A. Modeling
Stroke in Mice: Focal Cortical Lesions by
Photothrombosis. J Vis Exp.
2021;(171):10.3791/62536. Published 2021
May 6. doi:10.3791/62536

Berkeley Pharma Tech Journal of Medicine | 51



32. Fluri F, Schuhmann MK, Kleinschnitz C.
Animal models of ischemic stroke and their
application in clinical research. Drug Des Devel
Ther. 2015;9:3445-3454. Published 2015 Jul 2.
doi:10.2147/DDDT.S56071

33. Hermann DM, Popa-Wagner A,
Kleinschnitz C, Doeppner TR. Animal models
of ischemic stroke and their impact on drug
discovery. Expert Opin Drug Discov.
2019;14(3):315-326.
doi:10.1080/17460441.2019.1573984

34. Li Y, Zhang J. Animal models of stroke.
Animal Model Exp Med. 2021;4(3):204-219.
Published 2021 Sep 15.
doi:10.1002/ame2.12179

35. Marr RA, Thomas RM, Peterson DA.
Insights into neurogenesis and aging: Potential
therapy for degenerative disease? Future
neurology. Published July 1, 2010. Accessed
March 27, 2022.

36. Genet N, Hirschi KK. Understanding
neural stem cell regulation in vivo and applying
the insights to cell therapy for strokes. Regen
Med. 2021;16(9):861-870.
doi:10.2217/rme-2021-0022

37. Bulcha JT, Wang Y, Ma H, Tai PWL, Gao
G. Viral vector platforms within the gene
therapy landscape. Signal Transduct Target
Ther. 2021;6(1):53. Published 2021 Feb 8.
doi:10.1038/s41392-021-00487-6

38. Chen YC, Ma NX, Pei ZF, et al. A
NeuroD1 AAV-Based Gene Therapy for
Functional Brain Repair after Ischemic Injury
through In Vivo Astrocyte-to-Neuron
Conversion. Mol Ther. 2020;28(1):217-234.
doi:10.1016/j.ymthe.2019.09.003

39. Wang D, Tai PWL, Gao G.
Adeno-associated virus vector as a platform for

gene therapy delivery. Nat Rev Drug Discov.
2019;18(5):358-378.
doi:10.1038/s41573-019-0012-9

40. Liu D, Zhu M, Zhang Y, Diao Y. Crossing
the blood-brain barrier with AAV vectors.
Metab Brain Dis. 2021;36(1):45-52.
doi:10.1007/s11011-020-00630-2

41. Chan KY, Jang MJ, Yoo BB, et al.
Engineered AAVs for e�cient noninvasive gene
delivery to the central and peripheral nervous
systems. Nat Neurosci. 2017;20(8):1172-1179.
doi:10.1038/nn.4593

42. Ooboshi H, Ibayashi S, Takada J, Yao H,
Kitazono T, Fujishima M.
Adenovirus-mediated gene transfer to ischemic
brain: ischemic �ow threshold for transgene
expression. Stroke. 2001;32(4):1043-1047.
doi:10.1161/01.str.32.4.1043

43. Lukashev AN, Zamyatnin AA Jr. Viral
Vectors for Gene Therapy: Current State and
Clinical Perspectives. Biochemistry (Mosc).
2016;81(7):700-708.
doi:10.1134/S0006297916070063

44. Parambi DGT, Alharbi KS, Kumar R, et al.
Gene Therapy Approach with an Emphasis on
Growth Factors: Theoretical and Clinical
Outcomes in Neurodegenerative Diseases. Mol
Neurobiol. 2022;59(1):191-233.
doi:10.1007/s12035-021-02555-y

45. Chen YH, Keiser MS, Davidson BL. Viral
Vectors for Gene Transfer. Curr Protoc Mouse
Biol. 2018;8(4):e58. doi:10.1002/cpmo.58

46. Zu H, Gao D. Non-viral Vectors in Gene
Therapy: Recent Development, Challenges,
and Prospects. AAPS J. 2021;23(4):78.
Published 2021 Jun 2.
doi:10.1208/s12248-021-00608-7

Berkeley Pharma Tech Journal of Medicine | 52



47. Xu D, Su Y, Xu Q, Huang T, Chen Z,
Zhang T. Uniform iron oxide nanoparticles
reduce the required amount of
polyethylenimine in the gene delivery to
mesenchymal stem cells. Nanotechnology.
2021;33(12):10.1088/1361-6528/ac4066.
Published 2021 Dec 24.
doi:10.1088/1361-6528/ac4066

48. Oh J, Lee MS, Jeong JH, Lee M.
Deoxycholic Acid-Conjugated
Polyethylenimine for Delivery of Heme
Oxygenase-1 Gene in Rat Ischemic Stroke
Model. J Pharm Sci. 2017;106(12):3524-3532.
doi:10.1016/j.xphs.2017.07.020

49. Sainz-Ramos M, Gallego I, Villate-Beitia I,
et al. How Far Are Non-Viral Vectors to Come
of Age and Reach Clinical Translation in Gene
Therapy?. Int J Mol Sci. 2021;22(14):7545.
Published 2021 Jul 14.
doi:10.3390/ijms22147545

50. Lorenzini L, Baldassarro VA, Stanzani A,
Giardino L. Nerve Growth Factor: The First
Molecule of the Neurotrophin Family. Adv Exp
Med Biol. 2021;1331:3-10.
doi:10.1007/978-3-030-74046-7_1

51. Hernández-Echeagaray E. Neurotrophin-3
modulates synaptic transmission. Vitam Horm.
2020;114:71-89.
doi:10.1016/bs.vh.2020.04.008

52. Larpthaveesarp A, Ferriero DM, Gonzalez
FF. Growth factors for the treatment of
ischemic brain injury (growth factor
treatment). Brain Sci. 2015;5(2):165-177.
Published 2015 Apr 30.
doi:10.3390/brainsci5020165

53. Houlton J, Abumaria N, Hinkley SFR,
Clarkson AN. Therapeutic Potential of
Neurotrophins for Repair After Brain Injury:
A Helping Hand From Biomaterials. Front

Neurosci. 2019;13:790. Published 2019 Aug 2.
doi:10.3389/fnins.2019.00790

54. Li X, Li F, Ling L, Li C, Zhong Y.
Intranasal administration of nerve growth
factor promotes angiogenesis via activation of
PI3K/Akt signaling following cerebral
infarction in rats. Am J Transl Res.
2018;10(11):3481-3492. Published 2018 Nov
15.

55. Luan X, Qiu H, Hong X, et al. High serum
nerve growth factor concentrations are
associated with good functional outcome at
3 months following acute ischemic stroke. Clin
Chim Acta. 2019;488:20-24.
doi:10.1016/j.cca.2018.10.030

56. Cao JY, Lin Y, Han YF, et al. Expression of
nerve growth factor carried by pseudotyped
lentivirus improves neuron survival and
cognitive functional recovery of post-ischemia
in rats. CNS Neurosci Ther.
2018;24(6):508-518. doi:10.1111/cns.12818

57. Zhu W, Cheng S, Xu G, et al. Intranasal
nerve growth factor enhances striatal
neurogenesis in adult rats with focal cerebral
ischemia. Drug Deliv. 2011;18(5):338-343.
doi:10.3109/10717544.2011.557785

58. Hassanzadeh P, Arbabi E, Atyabi F,
Dinarvand R. Nerve growth factor-carbon
nanotube complex exerts prolonged protective
e�ects in an in vitro model of ischemic stroke.
Life Sci. 2017;179:15-22.
doi:10.1016/j.lfs.2016.11.029

59. Feczkó T, Piiper A, Ansar S, et al.
Stimulating brain recovery after stroke using
theranostic albumin nanocarriers loaded with
nerve growth factor in combination therapy. J
Control Release. 2019;293:63-72.
doi:10.1016/j.jconrel.2018.11.017

Berkeley Pharma Tech Journal of Medicine | 53



60. Yang J, Wu S, Hou L, et al. Therapeutic
E�ects of Simultaneous Delivery of Nerve
Growth Factor mRNA and Protein via
Exosomes on Cerebral Ischemia. Mol Ther
Nucleic Acids. 2020;21:512-522.
doi:10.1016/j.omtn.2020.06.013

61. Rocco ML, Soligo M, Manni L, Aloe L.
Nerve Growth Factor: Early Studies and
Recent Clinical Trials. Curr Neuropharmacol.
2018;16(10):1455-1465.
doi:10.2174/1570159X16666180412092859

62. Gaidin SG, Turovskaya MV, Gavrish MS,
et al. The selective BDNF overexpression in
neurons protects neuroglial networks against
OGD and glutamate-induced excitotoxicity. Int
J Neurosci. 2020;130(4):363-383.
doi:10.1080/00207454.2019.1691205

63. Cunha C, Angelucci A, D'Antoni A, et al.
Brain-derived neurotrophic factor (BDNF)
overexpression in the forebrain results in
learning and memory impairments. Neurobiol
Dis. 2009;33(3):358-368.
doi:10.1016/j.nbd.2008.11.004

64. Chaturvedi P, Singh AK, Tiwari V, Thacker
AK. Brain-derived neurotrophic factor levels in
acute stroke and its clinical implications. Brain
Circ. 2020;6(3):185-190. Published 2020 Sep
30. doi:10.4103/bc.bc_23_20

65. Yu SJ, Tseng KY, Shen H, Harvey BK,
Airavaara M, Wang Y. Local administration of
AAV-BDNF to subventricular zone induces
functional recovery in stroke rats. PLoS One.
2013;8(12):e81750. Published 2013 Dec 2.
doi:10.1371/journal.pone.0081750

66. Chen HH, Zhang N, Li WY, et al.
Overexpression of brain-derived neurotrophic
factor in the hippocampus protects against
post-stroke depression. Neural Regen Res.
2015;10(9):1427-1432.

doi:10.4103/1673-5374.165510

67. Shih HC, Kuan YH, Shyu BC. Targeting
brain-derived neurotrophic factor in the medial
thalamus for the treatment of central
poststroke pain in a rodent model. Pain.
2017;158(7):1302-1313.
doi:10.1097/j.pain.0000000000000915

68. A controlled trial of recombinant
methionyl human BDNF in ALS: The BDNF
Study Group (Phase III). Neurology.
1999;52(7):1427-1433.
doi:10.1212/wnl.52.7.1427

69. de Miranda AS, de Barros JLVM, Teixeira
AL. Is neurotrophin-3 (NT-3): a potential
therapeutic target for depression and anxiety?.
Expert Opin Ther Targets.
2020;24(12):1225-1238.
doi:10.1080/14728222.2020.1846720

70. Duricki DA, Hutson TH, Kathe C, et al.
Delayed intramuscular human neurotrophin-3
improves recovery in adult and elderly rats after
stroke. Brain. 2016;139(Pt 1):259-275.
doi:10.1093/brain/awv341

71. Duricki DA, Drndarski S, Bernanos M, et
al. Stroke Recovery in Rats after
24-Hour-Delayed Intramuscular
Neurotrophin-3 Infusion. Ann Neurol.
2019;85(1):32-46. doi:10.1002/ana.25386

72. Kang SS, Keasey MP, Arnold SA, Reid R,
Geralds J, Hagg T. Endogenous CNTF
mediates stroke-induced adult CNS
neurogenesis in mice. Neurobiol Dis.
2013;49:68-78. doi:10.1016/j.nbd.2012.08.020

73. Kumon Y, Sakaki S, Watanabe H, et al.
Ciliary neurotrophic factor attenuates spatial
cognition impairment, cortical infarction and
thalamic degeneration in spontaneously
hypertensive rats with focal cerebral ischemia.

Berkeley Pharma Tech Journal of Medicine | 54



Neurosci Lett. 1996;206(2-3):141-144.
doi:10.1016/s0304-3940(96)12450-2

74. MacLaren RE, Buch PK, Smith AJ, et al.
CNTF gene transfer protects ganglion cells in
rat retinae undergoing focal injury and branch
vessel occlusion. Exp Eye Res.
2006;83(5):1118-1127.
doi:10.1016/j.exer.2006.05.019

75. Chew EY, Clemons TE, Ja�e GJ, et al.
E�ect of Ciliary Neurotrophic Factor on
Retinal Neurodegeneration in Patients with
Macular Telangiectasia Type 2: A Randomized
Clinical Trial. Ophthalmology.
2019;126(4):540-549.
doi:10.1016/j.ophtha.2018.09.041

76. Xu B, Xie X. Neurotrophic factor control
of satiety and body weight. Nat Rev Neurosci.
2016;17(5):282-292. doi:10.1038/nrn.2016.24

77. Beker MÇ, Beker M, Çağlayan AB, et al.
Striatal dopaminergic neurons as a potential
target for GDNF based ischemic stroke therapy
[published online ahead of print, 2021 Nov
13]. Turk J Med Sci.
2021;10.3906/sag-2108-268.
doi:10.3906/sag-2108-268

78. Lohmann S, Grigoletto J, Bernis ME, et al.
Ischemic stroke causes Parkinson's disease-like
pathology and symptoms in transgenic mice
overexpressing alpha-synuclein. Acta
Neuropathol Commun. 2022;10(1):26.
Published 2022 Feb 24.
doi:10.1186/s40478-022-01327-6

79. Beker M, Caglayan AB, Beker MC, et al.
Lentivirally administered glial cell line-derived
neurotrophic factor promotes post-ischemic
neurological recovery, brain remodeling and
contralesional pyramidal tract plasticity by
regulating axonal growth inhibitors and
guidance proteins. Exp Neurol.

2020;331:113364.
doi:10.1016/j.expneurol.2020.113364

80. Gash DM, Gerhardt GA, Bradley LH,
Wagner R, Slevin JT. GDNF clinical trials for
Parkinson's disease: a critical human
dimension. Cell Tissue Res. 2020;382(1):65-70.
doi:10.1007/s00441-020-03269-8

81. Zhu W, Fan Y, Hao Q, et al. Postischemic
IGF-1 gene transfer promotes neurovascular
regeneration after experimental stroke. J Cereb
Blood Flow Metab. 2009;29(9):1528-1537.
doi:10.1038/jcbfm.2009.75

82. Okoreeh AK, Bake S, Sohrabji F.
Astrocyte-speci�c insulin-like growth factor-1
gene transfer in aging female rats improves
stroke outcomes. Glia. 2017;65(7):1043-1058.
doi:10.1002/glia.23142

83. Wang YQ, Guo X, Qiu MH, Feng XY, Sun
FY. VEGF overexpression enhances striatal
neurogenesis in brain of adult rat after a
transient middle cerebral artery occlusion. J
Neurosci Res. 2007;85(1):73-82.
doi:10.1002/jnr.21091

84. Markosyan V, Sa�ullov Z, Izmailov A, et al.
Preventive Triple Gene Therapy Reduces the
Negative Consequences of Ischemia-Induced
Brain Injury after Modelling Stroke in a Rat.
Int J Mol Sci. 2020;21(18):6858. Published
2020 Sep 18. doi:10.3390/ijms21186858

85. Shen F, Fan Y, Su H, et al. Adeno-associated
viral vector-mediated hypoxia-regulated VEGF
gene transfer promotes angiogenesis following
focal cerebral ischemia in mice. Gene Ther.
2008;15(1):30-39. doi:10.1038/sj.gt.3303048

86. Guo Z, Zhang L, Wu Z, Chen Y, Wang F,
Chen G. In vivo direct reprogramming of
reactive glial cells into functional neurons after
brain injury and in an Alzheimer's disease

Berkeley Pharma Tech Journal of Medicine | 55



model. Cell Stem Cell. 2014;14(2):188-202.
doi:10.1016/j.stem.2013.12.001

87. Pataskar A, Jung J, Smialowski P, et al.
NeuroD1 reprograms chromatin and
transcription factor landscapes to induce the
neuronal program. EMBO J. 2016;35(1):24-45.
doi:10.15252/embj.201591206

88. Jiang MQ, Yu SP, Wei ZZ, et al. Conversion
of Reactive Astrocytes to Induced Neurons
Enhances Neuronal Repair and Functional
Recovery After Ischemic Stroke. Front Aging
Neurosci. 2021;13:612856. Published 2021 Mar
26. doi:10.3389/fnagi.2021.612856

89. Ge L-J, Yang F-H, Li W, et al. In vivo
neuroregeneration to treat ischemic stroke
through neurod1 AAV-based gene therapy in
adult non-human primates. Frontiers in Cell
and Developmental Biology. 2020;8.
doi:10.3389/fcell.2020.590008

90. Chen YC, Ma NX, Pei ZF, et al. A
NeuroD1 AAV-Based Gene Therapy for
Functional Brain Repair after Ischemic Injury
through In Vivo Astrocyte-to-Neuron
Conversion. Mol Ther. 2020;28(1):217-234.
doi:10.1016/j.ymthe.2019.09.003

91. Mathiesen SN, Lock JL, Schoderboeck L,
Abraham WC, Hughes SM. CNS
Transduction Bene�ts of AAV-PHP.eB over
AAV9 Are Dependent on Administration
Route and Mouse Strain. Mol Ther Methods
Clin Dev. 2020;19:447-458. Published 2020
Oct 20. doi:10.1016/j.omtm.2020.10.011

92. Morales L, Gambhir Y, Bennett J, Stedman
HH. Broader Implications of Progressive Liver
Dysfunction and Lethal Sepsis in Two Boys
following Systemic High-Dose AAV. Mol Ther.
2020;28(8):1753-1755.
doi:10.1016/j.ymthe.2020.07.009

93. Gessler DJ, Tai PWL, Li J, Gao G.
Intravenous Infusion of AAV for Widespread
Gene Delivery to the Nervous System. Methods
Mol Biol. 2019;1950:143-163.
doi:10.1007/978-1-4939-9139-6_8

94. Fu H, McCarty DM. Crossing the
blood-brain-barrier with viral vectors. Curr
Opin Virol. 2016;21:87-92.
doi:10.1016/j.coviro.2016.08.006

95. Lochhead JJ, Thorne RG. Intranasal
delivery of biologics to the central nervous
system. Adv Drug Deliv Rev.
2012;64(7):614-628.
doi:10.1016/j.addr.2011.11.002

96. Finneran DJ, Njoku IP, Flores-Pazarin D, et
al. Toward Development of Neuron Speci�c
Transduction After Systemic Delivery of Viral
Vectors. Front Neurol. 2021;12:685802.
Published 2021 Aug 26.
doi:10.3389/fneur.2021.685802

97. O'Carroll SJ, Cook WH, Young D. AAV
Targeting of Glial Cell Types in the Central and
Peripheral Nervous System and Relevance to
Human Gene Therapy. Front Mol Neurosci.
2021;13:618020. Published 2021 Jan 11.
doi:10.3389/fnmol.2020.618020

98. Taschenberger G, Tereshchenko J, Kügler S.
A MicroRNA124 Target Sequence Restores
Astrocyte Speci�city of gfaABC1D-Driven
Transgene Expression in AAV-Mediated Gene
Transfer. Mol Ther Nucleic Acids.
2017;8:13-25. doi:10.1016/j.omtn.2017.03.009

99. Kumar A, Aakriti, Gupta V. A review on
animal models of stroke: An update. Brain Res
Bull. 2016;122:35-44.
doi:10.1016/j.brainresbull.2016.02.016

100. Ingberg E, Dock H, Theodorsson E,
Theodorsson A, Ström JO. E�ect of laser

Berkeley Pharma Tech Journal of Medicine | 56



Doppler �owmetry and occlusion time on
outcome variability and mortality in rat middle
cerebral artery occlusion: inconclusive results.
BMC Neurosci. 2018;19(1):24. Published 2018
Apr 19. doi:10.1186/s12868-018-0425-0

101. Cuccione E, Versace A, Cho TH, et al.
Multi-site laser Doppler �owmetry for assessing
collateral �ow in experimental ischemic stroke:
Validation of outcome prediction with acute
MRI. J Cereb Blood Flow Metab.
2017;37(6):2159-2170.

102. Ng YS, Stein J, Ning M, Black-Scha�er
RM. Comparison of clinical characteristics and
functional outcomes of ischemic stroke in
di�erent vascular territories. Stroke.
2007;38(8):2309-2314.
doi:10.1161/STROKEAHA.106.475483

103. Gri�n N, Faulkner S, Jobling P,
Hondermarck H. Targeting neurotrophin
signaling in cancer: The renaissance. Pharmacol
Res. 2018;135:12-17.
doi:10.1016/j.phrs.2018.07.019

104. Radin DP, Patel P. BDNF: An Oncogene
or Tumor Suppressor?. Anticancer Res.
2017;37(8):3983-3990.
doi:10.21873/anticanres.11783

105. Ikematsu Y, Tanaka K, Toyokawa G, et al.
NEUROD1 is highly expressed in
extensive-disease small cell lung cancer and
promotes tumor cell migration. Lung Cancer.
2020;146:97-104.
doi:10.1016/j.lungcan.2020.05.012

106. Li Z, He Y, Li Y, et al. NeuroD1 promotes
tumor cell proliferation and tumorigenesis by
directly activating the pentose phosphate
pathway in colorectal carcinoma. Oncogene.
2021;40(50):6736-6747.
doi:10.1038/s41388-021-02063-2

107. Disouky A, Lazarov O. Adult
hippocampal neurogenesis in Alzheimer's
disease. Prog Mol Biol Transl Sci.
2021;177:137-156.
doi:10.1016/bs.pmbts.2020.09.00

Berkeley Pharma Tech Journal of Medicine | 57


	Front_Page_Bhagwat
	Bhagwat_Galley
	references



