Multiplex genome modifications of astrocytes through CRISPR dead Cas۹: A novel candidate therapy for chronic ischemic stroke

Background and Aim: Stroke is an enervating injury to the brain, occurred from a stoppage in blood supply (ischemic stroke), or bleeding (hemorrhagic) in a hemisphere of the brain. Globally, about ۱۰ million deaths per year are recorded because of stroke. There has been no definitive FDA-approved treatment for chronic ischemic stroke without any side effects so far. Therefore, the search for new therapies is necessary. In this paper, we hypothesized improving the complications of chronic ischemic stroke in induced Sprague-Dawley rat model by intraluminal suture middle cerebral artery occlusion (MCAo), utilizing the combination of cell therapy and gene therapy. A new version of astrocytes is proposed by making some changes in their genome. To gain this goal, a gene profile including IL-۳۸ (the most modern anti-inflammatory agent, which barricades inflammatory response factors), BRAG-۱ (an anti-apoptotic gene from BCL-۲ family), IL-۳۸ and BRAG-۱’s complementary scaffold RNAs for their expression by deadCas۹ (dCas۹), complementary scaffold RNAs of LZK and MST-۱ for their deletion, and deadCas۹ gene is used. We hypothesized using modified astrocytes by dCas۹, which is the most accurate genome-editing technology with the least side effects, in improving the complications of chronic ischemic stroke.Methods: Searching online on google scholar, PubMed, and Scopus based on the keywords including Chronic ischemic stroke, Gene therapy, CRISPR, Scaffold RNAs, and Cell therapy.Results: The global occurrence level of stroke seemed to be steady between۱۹۹۰-۲۰۱۰, whereas there are some gain of ۶۸, ۸۴, ۱۲, and ۲۶%, in orderly in the occurrence of primary stroke, an outbreak of the stroke disability-adjusted lifespan lost, and the mortality rate of stroke. As mentioned before, stroke is the second cause of death universally, and there is no FDA-approved therapy and medication without any side effects for chronic ischemic stroke; for acute ischemic stroke, there is only one FDA-approved therapy, tissue plasminogen activator (tPA) with many side effects. So, research for a novel and safe therapy for chronic ischemic stroke is significant. In this hypothesis, we appraise the effects of transducing the gene profile into the ex vivo astrocytes from the brain of Sprague-Dawley rat models before induction of chronic ischemic stroke in them by Intraluminal suture middle cerebral artery occlusion (MCAo). The gene profile consists of IL-۳۸ as an anti-inflammatory agent, BRAG-۱ as an anti-apoptotic factor, IL-۳۸, and BRAG-۱’s complementary scaffold RNAs for their expression by dCas۹, complementary scaffold RNAs of LZK and MST-۱ for their deletion, and deadCas۹ gene. Leucine zipper-bearing kinase (LZK) has a crucial role in glial scar formation, while macrophage-stimulating ۱ (MST-۱) has a crucial role in the pathophysiological process of various neurological disorders and oxidative stress-induced neuronal cell decease. One problem in amending the deficits of chronic ischemic stroke is inflammation. Although astrocytes have an immunomodulatory function, IL-۳۸ is transfected into ex vivo astrocytes to augment their anti-inflammatory action, which is the newest anti-inflammatory agent from the IL-۱ family. It acts like IL-۱ receptor antagonist (IL-۱Ra) and IL-۳۶Ra, which prevents the production of T-cell cytokines like IL-۱۷, IL-۲۲, and IL-۸; so, it barricades inflammatory responses. LZK is a conserved mitogen-activated protein kinase kinase kinase (MAPKKK) upstream of c-Jun N-terminal kinase (JNK) in the mitogen-activated protein kinase (MAPK) pathway. It has been reported that the omission of LZK in adult mice astrocytes decreases astrogliosis and prevents scar formation in spinal cord injuries, so, by LZK deletion in astrocytes, their glial scar formation function could be inhibited. Also, based on the brain environment, astrocytes have different functions. Immediately after the stroke, they accumulate around the ischemic site to confine its propagation, repairing the blood-brain barrier, and confining the spread of inflammation and brain damage by producing a glial scar. Even though glial scar acts as a physical barrier and releases chondroitin sulfate proteoglycans (CSPGs), which prevents axon growth and regeneration, the recent pieces of evidence have demonstrated that glial scar helps in axon growth, but prevents the natural migration of NSCs. However, after stroke, astrocytes act differently. They have a significant role in fibrin devastation in CNS by preparing a surface for tissue plasminogen activator, which stimulates pro-brain-derived neurotrophic factor (pro-BDNF) and fibrinogen devastation, which prepares the ischemic area for the natural migration of NSCs. Another problem in the ischemic area is hypoxic death due to lack of circulation below ۵۰%, which results in the primary decrease of migrant NSCs, injected cells, and their derived cells. For preventing this hurdle, BRAG-۱ (an anti-apoptotic agent from the BCL۲ family) is transduced to astrocytes and MST-۱, necessary for oxidative stress-induced neuronal cell death, is inhibited. Moreover, pieces of evidence showed that specific deletion of MST-۱ in microglia relieves stroke-induced brain injury. Besides, astrocytes have a crucial role in controlling circulation and angiogenesis. This simultaneous expression and inhibition of these genes are just available by the newest, the most precise, cost-effective, and the easiest genome editing tool deadCas۹ and complementary scaffold RNAs of each gene. Another advantage of the CRISPR system is the possibility of switching the system off by anti-CRISPR proteins, whenever there are scarce side effects. As a result, a new version of astrocytes is produced, which their glia scar formation function is omitted, which makes the area ready for NSCs migration and following motor function improvement; they are resistant to hypoxic death and apoptosis; their anti-inflammatory role is amplified, and they have their angiogenesis function by releasing vascular endothelial growth factor (VEGF). Therefore, it is expected that they will be a novel multiplexed therapy of chronic ischemic stroke through a multiplex CRISPR system in SD rat models.Conclusion: The new version of astrocytes is proposed, which their glia scar formation function is omitted, which makes the area ready for NSCs migration and following motor function improvement. Besides, they are resistant to hypoxic death and apoptosis, their anti-inflammatory function is amplified, and they have their angiogenesis function by releasing VEGF. So, this could be the novel multiplexed therapy of chronic ischemic stroke in SD rat models, which has the potential to be used in the clinic, if further evaluation is performed.

Keywords:

Chronic ischemic stroke , Gene therapy , CRISPR , Scaffold RNAs , Cell therapy