�� 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. 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