Reneurish aims to genetically modify neural progenitor cells (NPC) to overexpress BDNF –a potent neurotrophic factor- to tackle the challenges attributed to neural transplants; low neuronal integration and cell survival. This overexpression is achieved through the infection of NPCs with an AAV vector. Donor’s NPCs are kept in a reservoir and infected after stroke takes place, then transplanted into the patient’s damaged brain region to enhance regeneration, and improve integration, synapsis and minimising tissue death. During our investigation, the need for precise monitoring led to the development of a customizable reporter transfection cassette, which can be easily removed during the humanization of the vector. This study concluded that BDNF can be overexpressed in human NPCs for over 50 days. We observed that an increase in BDNF levels correlates with a higher neural activity, better axon connectivity and higher cell count, without generating aberrant networks.
Every year, 15 million people suffer a stroke. From these patients, 5.5 million die, and 5 million survive but with permanent injuries [1]. This means that ischaemic stroke, representing 85 % of stroke cases, is the second leading cause of death in the world [2]. Not only that, but the risk of suffering a stroke is very significant, as the lifetime risk of suffering a stroke for people over the age of 25 is 25%.
The greatest stroke-derived burden is its side effects. Stroke accounts for almost 5% of all disability-adjusted life years (DALYs) in the world [3], which is strongly reflected on its devastating consequences, as it causes disability to millions of people every year. Its physical tolls suppose a disruption in the patients’ daily lives, which ends up being perjudicial to their mental health. Some of the physical side-effects can generate a decrease in visual field, body numbness, seizures, memory loss, aphasia, as well as spasms and loss of body movements. This leaves patients feeling as though they have no control over their own bodies, increasing their dependency and mental health alterations [4]. Neural lesions can alter the patients' emotion and behaviour, resulting in a higher risk of suffering from depression, anxiety, personality changes and impulsivity [5]. By 2030, ischemic stroke cases are projected to increase by 9.62 million [2], which will become a significant economical and social burden to countries with less resources. With stroke incidence on the rise, especially among the younger generations, the need for effective methods of rehabilitation is clear [6].
Current therapies for ischemic stroke are used to remove blood clots that are generated during the arterial blockage in the brain, and they are known as thrombolysis and thrombectomies [7]. Thrombolysis, with a functional window of 3 to 4.5 hours post-stroke [8], usually delivers tissue plasminogen activator (tPA) via an endovascular catheter. tPA is a protease enzyme that’s used to catalyse thrombolysis, helping dissolve platelet aggregates [9]. However, with its small functional window, the treatment is only suitable in 12 % of ischemic stroke cases [2]. Thrombectomy, on the other hand, has a functional window of 6 to 24 hours, and it is a mechanical approach to blood clot removal. Although it has more efficacy than intravenous thrombolytics like tPA [10], it’s still a fairly invasive procedure that can cause vessel perforation and dissection. Not only that, but the treatment is only applicable to 10 % of ischemic stroke cases.
Blood clot removal helps reduce disability post-stroke, but it cannot recuperate the necrotic loss of brain mass. To put it into perspective, after an untreated stroke, patients lose up to 2 million neurons per minute, making recovery especially gruelling. An option for neuronal recovery is via transplantation. For a transplant to be effective, the transplanted cells need to integrate into the receiving tissue, and form functional connections with pre-existing neurons [11]. Integration has proven to be a challenge, as low compatibility and lack of suitable cell types hinder their therapeutic use in this field [12].
As the need for neural injury rehabilitation treatments is not met in the market, we saw the opportunity to create a therapeutic alternative to restore lost neuron mass in ischaemic stroke patients. We propose a novel approach to neural transplants, by which the transplanted cells are transiently-modified neural progenitors (neural stem cells), enriched with neurotrophic factors. Neurotrophic factors are a type of neuropeptides that help regulate growth, differentiation and neural survival rates [13]. By enriching the transplanted cells with these factors, we hope to improve integration of the transplant into the receiving tissue, reducing neuronal necrosis, optimising new neural connections and minimising the side-effects of stroke, expediting recovery. After a thorough literature search, we have selected to express Brain-Derived Neurotrophic Factor (BDNF) with Neural Progenitor Cells (NPC), a well described molecule capable of repairing brain damage, with promising results in preclinical studies in mice [14].
BDNF does have drawbacks. Primarily, its short lifespan and difficulty to cross the blood-brain barrier make it unsuitable for bloodstream delivery. By expressing BDNF within neural tissue, we optimise its use to restore brain integrity and neuronal connections. Prolonged exposure to BDNF can have a certain degree of toxicity. To avoid this, we are controlling BDNF expression by making it time-dependent. This reduces production of BDNF to up to 3 months, minimising any possible negative effects. Another great factor that differentiates Reneurish from other therapies, is use of induced Pluripotent Stem Cell (iPSC)-derived NPC. Not only is the differentiation process extremely reliable and efficient, nullifying the over-proliferating state of stem cells and therefore avoiding formation of teratomas, but NPCs can differentiate into all kinds of neural cells, meaning patients can not only regain neuronal mass, but also glial cells, like astrocytes and oligodendrocytes [15]. By transfecting our progenitors with Adeno-Associated Viruses (AAV), whose genome does not integrate into the host cell, we ensure that the expression of BDNF will be temporary, and won’t be expressed in the long term [16]. Therefore, the final product we aim to design are neural progenitor cells genetically modified to express BDNF during a brief time period. What is also great about Reneurish is its future potential. As a universalised therapeutic, we open the possibility of treatment of many neuronal degenerative conditions, especially thanks to the enrichment of BDNF expression.
[1] WHO EMRO | Stroke, Cerebrovascular accident | Health topics. . [2] Projected Global Trends in Ischemic Stroke Incidence, Deaths and Disability-Adjusted Life Years From 2020 to 2030 | Stroke. https://www.ahajournals.org/doi/full/10.1161/STROKEAHA.122.040073?rfr_dat=cr_pub++0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org#. [3] Post-Stroke Rehabilitation | NINDS Catalog. https://catalog.ninds.nih.gov/publications/post-stroke-rehabilitation (2020). [4] Way of working. Stroke Data project https://www.strokedataproject.com/way-of-working/. [5] Ischaemic stroke | Stroke Association. https://www.stroke.org.uk/stroke/types/ischaemic. [6] Thrombolysis (Thrombolytic Therapy) for Blood Clots | Penn Medicine. https://www.pennmedicine.org/for-patients-and-visitors/find-a-program-or-service/heart-and-vascular/vascular-surgery-and-endovascular-therapy/vascular-procedures/thrombolysis. [7] Pan, Y. & Shi, G. Silver Jubilee of Stroke Thrombolysis With Alteplase: Evolution of the Therapeutic Window.Front. Neurol. 12, (2021). [8] Mathews, S. & De Jesus, O. Thrombectomy. in StatPearls (StatPearls Publishing, 2024). [9] Gage, F. H. & Temple, S. Neural stem cells: generating and regenerating the brain. Neuron 80, 588-601 (2013). [10] Kim, S. U. & de Vellis, J. Stem cell-based cell therapy in neurological diseases: a review. J Neurosci Res 87, 2183-2200 (2009). [11] Medical Definition of NEUROTROPHIC FACTOR. https://www.merriam-webster.com/medical/neurotrophic+factor. [12] Brain-Derived Neurotrophic Factor and Its Potential Therapeutic Role in Stroke Comorbidities - Liu - 2020 - Neural Plasticity - Wiley Online Library. https://onlinelibrary.wiley.com/doi/10.1155/2020/1969482. [13] Martínez-Cerdeño, V. & Noctor, S. C. Neural Progenitor Cell Terminology. Front Neuroanat 12, 104 (2018). [14] Wright, J. F. Transient Transfection Methods for Clinical Adeno-Associated Viral Vector Production. Hum Gene Ther 20, 698-706 (2009).