The vertebrate nervous system is comprised of two main parts: the central nervous system (CNS) consisting of the brain and spinal cord, and the peripheral nervous system (PNS) consisting of the neurons that connect the CNS to the rest of the body. These components collectively control all functions of the body, memory, mood, and senses, including pain.
Information on more than 1000 neurological diseases or disorders is searchable on an NIH website. According to a comprehensive statistical analysis, 100 million Americans are effected by at least one neurological conditions at some point during their life, with the five most burdensome being stroke, Alzheimer’s/other dementias, migraine, epilepsies, and Parkinson’s.
The viral vector gene therapy field for neurological diseases was recently reviewed (Morris & Schorge, 2022). These authors suggest that alternative mRNA approaches for neurological diseases are attractive because they are non-permanent, controllable, and therefore inherently safer than viral vectors.
This blog discusses recent findings from investigations that utilize TriLink products while investigating mRNA therapeutic development for five neurological conditions:
- Spinal Cord Injury
- Spinal Disc Degenerative Disease
- Niemann-Pick Disease
- Peripheral Neuropathy
According to NIH statistics, nearly 800,000 Americans have a stroke each year. A stroke is defined as a sudden interruption of blood flow to the brain when a blood vessel in the brain becomes blocked or bursts. As the most burdensome neurological disease both in the US and globally, stroke is a prime target for developing mRNA therapeutics.
Neuronal cell death can be caused by inadequate blood supply (ischemia) and can lead to cardiac arrest and lifelong neurological deficits. Currently, there are no effective treatments for neuronal death because of the vulnerability of the neurons to death. Symptoms can be mitigated by reopening the occluded artery and cardiopulmonary resuscitation within a very brief time.
Because brain-derived neurotrophic factor (BDNF) expression is upregulated after nerve injury, administration of recombinant BDNF protein has been investigated to prevent neuronal death, albeit unsuccessfully so far. Fukushima et al. (2021) hypothesized this failure might be due to non-penetration of the blood-brain barrier by BDNF and its short half-life, both of which could be mitigated by local delivery of encapsulated BDNF mRNA.
In a rat model of transient global ischemia, intraventricular administration of BDNF mRNA using a polymer-based carrier increased the survival rate of hippocampal neurons with a rapid rise of BDNF levels. Remarkably, dosing on days 2 and 5 after transient global ischemia led to therapeutic improvements in spatial memory compared with untreated controls on day 20.
A related study by Xu et al. (2022) used TriLink Cas9 mRNA for CRISPR editing to obtain G protein-coupled receptor 39 (GPR39) knock-out mice to show that, after cerebral ischemia, absence of GPR39 worsens capillary blood flow and exacerbates brain injury and functional deficit. These new findings led to the suggestion that the known (Xie et al. 2021) activation of GPR39 function by a synthetic chemical agonist may provide a protective treatment for ischemic stroke.
2. Spinal Cord Injury
Traumatic spinal cord injury (SCI) affected nearly 300,000 Americans in 2021, according to available statistics. Within days of the primary injury, neurons and glia cells in the lesion area die, resulting in functional deficits. Although viral vector administration of interleukin-10 (IL-10) protein has shown promise in functional recovery from SCI in a mouse model (Smith et al. 2020), there are safety concerns for use of such vectors. Recently, Weissman & collaborators reported an investigation of IL-10 mRNA as a more controllable and safer approach for treating SCI.
Briefly, the researchers first measured the distribution and duration of enhanced green fluorescent protein (eGFP) expression in the CNS following intraspinal injection in rats. Using a lipid nanoparticle (LNP) formulation of CleanCap® N1-methylpseudouridine (m1ψ) eGFP mRNA, synthesized with TriLink reagents, a single injection led to strong fluorescence at the injection site for 5 days, followed by gradual diminution up to 21 days.
The same dose of a LNP formulated CleanCap® m1ψ-modified IL-10 mRNA, synthesized with TriLink reagents, was administrated 1 week after contusion-induced SCI in rats. This treatment, which led to IL-10 expression distribution and duration comparable to that for eGFP, induced significant morphological and functional recovery of the injured spinal cord tissue. A standard locomotor rating scale and a custom-designed kinematic videography method both showed continuous motor improvement up to 9 weeks after dosing compared to untreated controls.
3. Spinal Disc Degenerative Disease
Spinal disc degenerative disease (DDD) leads to lower back and neck pain and remains one of the leading causes of disability across the globe (Chang et al., 2022). The spinal disc is comprised of a central hydrophilic proteoglycan-rich gelatinous core that absorbs compressive force. Maintenance of this core’s extracellular matrix is normally homeostatic, but excessive stresses or other stimuli can alter this homeostasis and initiate DDD.
To investigate the possibility of a mRNA therapeutic for DDD, Chang et al. focused on the protein transcription factor termed Runx1, which is involved in stimulation of the extracellular matrix in damaged discs and expression of collagen. For feasibility of delivery, luciferase (Luc) mRNA either complexed with a synthetic polymer or naked were injected locally in a rat model of DDD. In vivo bioluminescence imaging showed that the complexed Luc mRNA was expressed for up to 6-days post-injection while naked Luc mRNA was expressed for only 6-hours post-injection.
Naked and complexed Runx1 mRNA were each again injected in this DDD model and in vivo MRI was used to quantitatively measure disc hydration content, an essential indicator of disc health status. Only sparse water content was found 2-weeks and 4-weeks post-injection with naked Runx1 mRNA, whereas 43% and 104% more water were found for complexed Runx1 at these time points. Thus, this study suggests further studies of Runx1 mRNA treatment for DDD are warranted.
4. Niemann-Pick Disease (NPD)
As reviewed elsewhere, Niemann-Pick disease is an inherited neurological condition involving accumulation of unesterified cholesterol and glycosphingolipids in cells throughout the body, resulting in damage to the CNS. The neurological dysfunction, which has various manifestations, is progressive and invariably fatal. NPD arises from mutations in the gene for a protein termed NPC, which normally transports unesterified cholesterol across the endosomal membrane into the cytosol. There is no approved therapy of NPD, only treatment of symptoms.
Furtado et al. (2022) investigated whether mRNA treatment could correct the biochemical and phenotypic hallmarks of NPD in vitro. Toward this end, extensive codon optimization, nucleoside modification, and mRNA melting studies of secondary structure were first carried out with a Luc mRNA reporter. Modified nucleosides included m1ψ, 5-methoxyuridine (5-moU), and 5-methylcytosine (m5C), all of which are available from TriLink as 5’-triphosphate reagents. Luc expression in three different cell lines was monitored following transfection with Lipofectamine, with the found order of Luc expression being m1ψ > 5-moU > m5C > unmodified.
They next investigated whether similarly engineered NPC mRNA variants (with m1ψ, 5-moU or unmodified) restored functional protein expression in NPD patient fibroblasts in vitro. At 24-hr post-transfection, all three mRNAs generated substantial NPC protein expression; however, further work was focused only on m1ψ-modified NPC mRNA.
They continued their studies to better understand if the mRNA treatments could reverse disease pathology. NPC m1ψ-mRNA treatment restored the cholesterol esterification capacity of patient cells to wildtype levels, and significantly reduced both unesterified cholesterol levels (>57%) and lysosome size compared to untransfected controls. This first-time demonstration of the use of mRNA to rescue the NPC protein insufficiency and pathogenic NPD phenotype in vitro sets the stage for future in vivo studies.
5. Peripheral Neuropathy
Peripheral neuropathy caused by peripheral nerve damage is a common medical condition causing pain, tingling, or numbness in affected areas (Yu et al., 2023). This damage has various causes, including diabetes, traumatic injury, genetics, and chemotherapy. Notably, chemotherapy-induced peripheral neuropathy is a dose-limiting side-effect of chemotherapy regimens in 80%-90% of cancer patients.
Nerve growth factor (NGF) protein promotes survival, proliferation, and neurite outgrowth of neurons. Recombinant NGF supplementation is an emerging therapeutic strategy for neuronal damage. However, the clinical efficacy of NGF is limited by its short half-life and its facilitating pain-processing, which hamper its clinical application. Therefore, a “painless” form of NGF with a prolonged-expression pattern would be ideal for nerve regeneration.
Serendipitously, it was previously discovered (Testa et al. 2019) that persons afflicted with hereditary sensory and autonomic neuropathy type V have loss of pain sensation resulting from a “painless” mutant form of NGF (NGFMUT). Since NGFMUT is highly glycosylated, it is not amenable to recombinant protein production. Therefore, Yu et al. investigated NGFMUT mRNA as a potential therapeutic for peripheral neuropathy.
To begin their work, Yu et al. codon-optimized a m1ψ-substituted mRNA encoding either NGFMUT or wild-type NGF (NGFWT). They used these mRNAs to assess protein expression and secretion following in vitro transfection into [XX cell system]. Expression of NGFMUT was robust, but its secretion was lower than NGFWT. Consequently, several alternative leader peptide sequences for NGFMUT were tested, leading to use of Ig Kappa signal peptide (IGK-NGFMUT) to maintain expression and improve secretion levels.
Yu et al. next showed that administration of IGK-NGFMUT-mRNA-LNP to mice led to significantly lower responses to either mechanical or thermal stimuli, compared to control mice administered NGFWT-mRNA-LNP. Finally, IGK-NGFMUT-mRNA-LNP was studied in a cancer drug (paclitaxel)-induced peripheral neuropathy mouse model. Mice were administered paclitaxel followed by five local injections of IGK-NGFMUT-mRNA-LNPs into the hind paw every three days. The IGK-NGFMUT-mRNA-LNP treatment significantly lessened sensitivity to thermal stimuli compared to control treatments, thus indicating possible clinical utility.
The above examples of mRNA treatments for pain and neurological diseases demonstrate the seemingly ever-widening potential scope of therapeutic applications of synthetic mRNA. mRNA’s safety, specificity, versatility, and manufacturability advantages continue to allure researchers to test its potential in their experimental systems.
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