Renewal: SynBio solutions to Neurodegenerative Disease

One of the most terrifying ailments is the loss of the self. The loss of memory, perception, and awareness. These are integral to the living experience of any thinking being. In scientific terms, such ailments are termed Neurodegenerative Diseases that include a wide range of symptoms like loss of cognitive function, reduction in memory formation and retention, neuron loss, degradation of motor coordination and increased tremors.

Neurodegenerative Disorders/Diseases (NDs) refer to the impairment of synapses, neuron networks or the deposition of various altered proteins: often used as the identifying factor (Amyloid-Beta and Tau plaques in Alzheimer’s, alpha-synuclein in Parkinson’s and TAR DNA-binding protein in ALS). Largely, these proteins disrupt biochemical activity and damage neural structures by degradation or detrimental enlargement. Protein misfolding and genetic defects are so far the major factors which are linked with NDs.

Neurodegenerative disorders are among the oldest and yet least resolved. The brain, as it turns out, is terrible at introspection. Fairly reasonable too: it’s the most complex machine humans have ever encountered, with 10 times as many neurons in a single unit as there are humans on this planet. Historically, the goal of neuroscience was not to treat NDs but to understand them. “People going into neurology were always warned that it was a specialty in which one could diagnose, but not treat.” [5]. The primary difficulty was the lack of a comprehensive and efficient method of studying the living brain. “The current gold standard for diagnosing neurodegenerative disorders is neuropathological evaluation at autopsy.” [3]. That’s certainly a chilling statement, denoting that the best diagnostic process (as of then) was poking around the nervous system after death.

This was changed by the advent of novel imaging and biochemical tracing technologies (such as kainate injections combined with the Fink-Heimer technique). Soon, through analysis of animal brain lesions and the imaging of neurons, axons and minute neural structures, impressive strides were made in our understanding of the brain. Focus on neurotransmitters soon revealed that multiple NDs were linked to a decrease in specific neurotransmitter levels, markers and receptors (dopaminergic cells and acetylcholine in Alzheimer’s, GABAergic markers in Huntington’s). Further research in the ’70s uncovered the role of genetic mutations and multiple misfolded proteins leading to different kinds of dysfunctions in the brain (such as Amyloid-Beta plaques and neurofibrillary tangles of misfolded Tau protein).

NDs are classified then by various parameters such as :

1. Primary clinical features (Dementia, motor neuron disease, and parkinsonism)
2. Anatomy of neurodegeneration (frontotemporal degeneration, extrapyramidal disorders or spinocerebellar degenerations)
3. Principal molecular abnormality (amyloidoses, tauopathies, alpha-synucleinopathies and TDP-43 proteinopathies)

The next stage in pathology after images and chemical tracers is clearly a direct study of the brain tissue itself. Live human brains of course cannot be studied without following up with an impromptu autopsy. Thus, modelling the human brain is of paramount importance to neuroscience. There are two approaches to this: a digital neural imitation (which is still far away from a complete mapping), or a biological model: something that is more physical and possible in the present day. Here is where synthetic biology jumps in.

Animal models are useful analogues for human pathology, but they are limited in the sense that they do not faithfully represent all the aspects of human tissue, are often restricted by environmental factors and give misleading results that may not always be recreated in humans. To circumvent this issue, organoids created using iPSCs directly from the patient’s cell line are immensely valuable. Induced Pluripotent Stem Cells (iPSCs) are created by reprogramming highly differentiated cells back to the pluripotent “Stem Cell” age. These can then be grown into imitations of any cell in the body, and thus an in vitro tissue model for the brain. Cerebral organoids are structures of remarkable complexity, and can help model protein aggregation, drug response and study selective vulnerability. Some major drawbacks include a lack of cell diversity, vasculature and controlled heterogeneity, which are possible future directions for comprehensive ND modelling.

But that’s only about diagnosis. A major hurdle that neuroscience research faces today is a lack of effective treatment plans for many common neurodegenerative disorders, which is again due to the dearth of pathological understanding and highly variable environmental and genetic causes for the same illness. However, aimed specifically at the genetic causes of NDs, there are multiple hopeful solutions.

Genes directly or indirectly control the formation of neural structures, expression of physiological features and hormone distribution. Damaged genes can then hamper all of these by reducing the production of an essential neurotransmitter or by the accumulation of unfolded proteins in a vital area leading to fallout over the metabolic activity of the entire brain. Why not repair or replace such damaged genes? Gene therapy is the fundamental solution to this question.

The standard procedure is to infuse modified non-replicative adeno-viral (or lentivirus) modified gene vectors, which can deliver healthy genes or gene modulating factors to the relevant tissue. These can be intelligently customised to the specific cell subtype that is being targeted. Gene therapy is especially effective in monogenic diseases wherein the target gene is known, understood and can be rectified individually. However, even single-gene factors are not simple when embedded in disease-causing networks, so a safer strategy is that of neuroprotection: delivering neurotrophic factors which promote cell growth and maintenance, or cells which themselves secrete neurotrophic factors embedded in the neural tissue.

Synthetic biology tools are available even for genetic payload design, harbouring the potential to help engineer safe and effective transcription factors that control gene expression. RNA silencing/editing has even had successful pre-clinical trials. Mutated RNA transcripts can be silenced using RNAi (RNA interference) molecules. The use of modified small hairpin oligonucleotides (shRNAs) or microRNAs (miRNAs) has shown results in a safe and long-term treatment of Huntington’s Disease in a non-human primate. Clinical trials have been designed for oligonucleotides that target Spinal Muscular Atrophy. Dangers in this technique are mainly related to the immune system and cellular enzyme response to the modified polymers. Synthetic biology technologies can help expedite the biomanufacturing of novel, entirely synthetic polymers that are not degraded by cellular enzymes. Artificial proteins can even be fused to activating transcription factors, which work towards upregulating endogenous neurotrophic factors. Thus, there are extensive possibilities for specified artificial gene regulation.

Synthetic Nuclease based genome editing provides us an extremely powerful avenue for ND gene therapy. CRISPR/Cas9 systems have flourished in recent years, providing opportunities for efficient, extensive and easy genetic modification of mammalian cells. We can entirely remove, modify or replace a defective disease-causing gene with great ease. Again, pluripotent stem cells provide a valuable approach.

Lysosomal storage diseases are characterised by neurodegeneration due to build-up of lysosome by-products. A 2013 clinical trial successfully used the transplantation of engineered haematopoietic cells which can migrate to the brain and clear the toxic accumulations of the enzyme substrate. Monogenic metabolic NDs are ripe for ex vivo treatment: immune rejection is not an issue and editing factors can be delivered as external RNA, reducing awry targets and long-term expression. Similarly, selective monoclonal antibodies can be applied to clear accumulated proteins.

These treatment plans are all developed externally and delivered. Gene circuits facilitate intelligent, adaptive networks which can precisely control the expression of proteins. External gene therapy vectors are often limited, utilizing finished therapeutic proteins expressed by simple promoters. Gene switches (small molecules or proteins such as mifepristone) are utilized for conditional expression (preventing overregulation and out-of-control production of proteins).

Combining all of these technologies, we can create synthetic cells equipped with gene circuits encapsulated within a semipermeable material that are used to release therapeutic compounds or factors from within the body. Several instances of clinical trials in interesting albeit limited contexts have been worked upon already: encapsulated cells that release neurotrophic factors ([12]), double treatment utilizing amine receptor linked to G proteins targeting metabolic syndromes ([13]), and biosensors linked to dopamine receptors ([14] [15]).

While not a conquered battlefield, NDs are under the onslaught of meticulous and expanding research in neuroscience and synthetic biology. The coming decades are sure to provide us victories that steal a few more lives from the clutches of disease.

References:

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