Project Description

How it all started?

Our team devoted the past two years to the diagnosis and treatment of two different forms of cancer; however, this year we decided to explore a whole new fascinating field: treating the world's most severe neurodegenerative disease. It is time to harness the remarkable capabilities of Synthetic Biology to help over 35 million people worldwide remember again.

Our project, named "Lethe", represents an innovative approach in the world of Alzheimer's disease treatment, one of the most significant public health challenges globally. Looking ahead, our vision extends beyond initial milestones and addresses the rapid and anticipated increase in the number of affected individuals, expected to triple by 2050.

Why Lethe?

In Greek mythology, Lethe was one of the five rivers of the underworld, Hades, whose name means "oblivion" or "forgetfulness". Souls who drank from Lethe's waters would forget their earthly lives entirely before moving on to the afterlife. This image of profound forgetfulness resonates with the experience of those suffering from Alzheimer's disease, where memories and identities slowly fade away.

Alzheimer's disease, like the waters of Lethe, erases the precious moments and connections that define us. With "Lethe", we turn the tide against this relentless river of forgetfulness. Through the power of Synthetic Biology, we develop a therapy that restores memories, helping individuals hold on to the stories that make life meaningful. By choosing the name Lethe, we remind ourselves of the importance of memory and our commitment to combat the devastating effects of this disease, offering hope and the promise of remembrance.


The problem

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia, ranking as the 7th leading cause of death globally.

AD is characterised by the accumulation of abnormal protein aggregates. The neuropathological hallmarks of the disease are plaques formed around deposits of extracellular amyloid beta (Aβ) proteins and endocellular neurofibrillary tangles (NFT) of accumulated hyperphosphorylated tau protein. Normally, tau protein stabilises axonal microtubules, essential for intracellular transport. Both of these pathological changes disrupt neuronal function and connectivity, ultimately leading to neuronal death and brain atrophy.

AD is primarily classified into two subtypes based on age of onset and genetic factors: sporadic Alzheimer's disease (SAD) and familial Alzheimer's disease (FAD). SAD is the most common form, accounting for approximately 95-99% of cases, typically occurring in individuals over the age of 65. FAD, which is much less common, represents about 1-5% of cases and usually manifests earlier in life, often before the age of 60, due to inherited mutations in specific genes, such as APP, PSEN1, and PSEN2.


Symptomatology and Stages

The symptoms of the disease vary according to the stage in which the patient belongs.
  • In the early stage, patients suffering from the disease experience gradual memory loss and increasing confusion, their ability to perceive space and time decreases, and the first changes in personality and behaviour appear.
  • Moving into the middle stage, patients experience a gradual loss of speech, empathy, consciousness, and other senses, as well as an inability to recognize familiar faces as memory loss accelerates. They also present a multitude of psychological problems and sleep disorder issues.
  • By reaching the final stage, Alzheimer's patients experience complete loss of communication, become solely dependent on others, and are vulnerable to infections as bodily functions progress towards death.

Our pharmaceutical approach against other pharmaceutical substances

The drugs on the market for Alzheimer's disease are divided into two categories: symptomatic drugs and therapeutic antibodies. The first category consists of acetylcholinesterase inhibitors and NMDA receptor antagonists whose approach is palliative and provides modest short-term benefits. The second category targets extracellular amyloid beta (Aβ) plaques but without efficiently fighting the disease, while having serious side effects. None of the available therapeutic agents effectively alter the underlying neurodegenerative process.

However, our pharmaceutical approach shows promise for the inhibition and treatment of Alzheimer's disease with minimal side effects. Our drug does not aim to inhibit the progression of disease symptoms, as existing drugs do, but directly targets the neuropathophysiological process responsible for its appearance and further development. Our approach focuses on tau proteins, which have not been extensively exploited pharmacologically but play a crucial role in the pathophysiology of the disease.

Solution

In a few words…

The "Lethe" project proposes a new therapeutic approach for the treatment and management of Alzheimer's Disease. By targeting the dephosphorylation of tau proteins, we increase the chances of preventing the disease and reversing the underlying neurodegenerative course. Our goal is to enhance the activity of the phosphatase PP2A, which is related to the process of tau dephosphorylation. This up-regulation will be facilitated indirectly by microRNA-195, a molecule associated with various pathological processes of the disease. We will introduce microRNA-195 into nerve cells using AAV-carrying exosomes, modified with the RVG29 peptide that binds specifically to target nerve cells. These modified exosomes will interact with the surface of the target cells and release AAVs expressing microRNA-195, inhibiting the processes responsible for the disease's pathogenesis.

Solution

The multi-hit action of microRNA-195 against Alzheimer's Disease


Micro-RNAs (miRNAs) are small, single-stranded, non-coding RNA molecules, typically 20-23 nucleotides long, found in animals, plants, and some viruses. Around 2,600 mature miRNAs are identified in humans, with about half located in gene introns and the other half in independent genes.

In the cytoplasm, miRNAs regulate gene expression by binding to the 3' UTR of mRNA, repressing translation or promoting degradation via deadenylation and decapping. Their action is controlled by cell-specific expression and compartmentalization. The crucial role micro-RNAs play in gene expression, their natural role as repressing regulatory molecules, and wide range of action, led us to choose them for our gene therapy model.

Our micro-RNA of choice is micro-RNA 195. Although initially used for its tumor suppressor qualities to fight cancer, it has been shown that this micro-RNA is strongly correlated to Alzheimer’s disease (AD). Either directly or indirectly, the downregulation of micro-RNA 195 is tied to increased AD pathology. More specifically, micro-RNA 195 has been shown to specifically suppress the expression of BACE-1 and APP proteins, which are both essential for the formation of Αβ – amyloid plaques, a key pathogenic feature of AD.

At the same time, it has been shown that downregulation of mir-195 is strongly linked with Tau protein hyperphosphorylation. This is due to the interplay between mir-195 and PP2A’s regulatory pathway.


Solution

PP2A is the chief phosphatase responsible for the dephosphorylation of Tau protein on a clinical level, which initially led us to turn our attention to this protein. Its dephosphorylation qualities hinted at the possibility that PP2A can actively fight and reverse Tau protein hyperphosphorylation, essentially targeting AD pathophysiology to its core.

But first, an understanding of PP2A’s regulation is necessary to appreciate the value of mir-195. PP2A achieves active form when it is methylated on residue Leukine 309 and dephosphorylated on residue Tyrosine 307. The former is controlled by two proteins, LCMT1 leucine carboxyl methyltransferase, which enables the methylation and PME1 protein phosphatase methylesterase 1, which removes the methylation. The phosphorylation on the other hand is made possible by GSK-3β kinase.

Mir-195’s role in this pathway is that it directly reduces the levels of PME-1 by binding to PME-1’s mRNA’s 3’ UTR, thus hindering the production of the protein on a post-transcriptional level. This way, Methylation of PP2A in Leu309 is increased. Mir-195 also, by reducing the Αβ – amyloid plaque formation, affects LCMT-1 and GSK-3β kinase. It has been shown that Αβ – amyloid plaque accumulation boosts the action of GSK-3β while it reduces the action of LCMT-1. Mir-195 thus, will have the opposite action in the AD afflicted brain, and as result increase methylation on Leu309 through LCMT-1 and reduce phosphorylation of Tyr307 through GSK-3β.

All this starkly hints at the ability of mir-195 to boost the action of PP2A and consequently alleviate Tau hyperphosphorylation.

Additionally, mir-195 reduces the phosphorylation of Tau without involving PP2A too. Tau is phosphorylated by GSK-3β and cdk5 kinases, both of whose action is enhanced by Αβ – amyloid plaque formation. Mir-195, by reducing the latter, also reduces the efficiency of these kinases. This way, the phosphorylation level of Tau is decreased.

Lastly, micro-RNA is shown to have anti-inflammatory properties. AD is always categorized as a neuroinflammatory disease, due to the inflammation caused by Αβ amyloid plaques and tau protein aggregates. To alleviate this inflammation is to fight the harm AD causes on the patient, and this is exactly what mir-195 does. Firstly, this is achieved in indirect fashion with the reduction of Αβ amyloid plaques, but also directly, through mir-195 decisive interplay in neuron-microglia communication. In short, mir-195 regulates the CX3CL1/CX3CR1 signaling cascade, a process responsible for the polarization of microglia cells to macrophages through the M1 pathway, resulting in increased inflammatory state of many regions in the brain . Mir-195 successfully diverts this polarization by acting on the CX3CL1/CX3CR1 pathway, and thus fighting against neuroinflammation.

Mir-195 is able to reverse neuronal loss, which is typical of the pathology of AD, and the most common reason for the worst of AD’s symptoms. Mir -195 plays a crucial role on the N-APP/DR6 pathway controlling neuronal apoptosis and neurite outgrowth. Specifically, N-APP, a precursor protein of Αβ amyloid plaques, binds to Death Receptor DR6 and induces neuronal apoptosis and degeneration of neurons alike. Mir-195 targets and reduces the levels of both of those proteins, effectively halting and reversing one of AD’s most lethal features.

This wealth of actions, render hsa-mir-195-5p as the essential microRNA for the brain in AD.

rAAV: an ideal tool for CNS gene therapy

The goal of this project is to construct an optimal and novel gene therapy delivery model for battling Alzheimer’s disease. To achieve this goal, a suitable vector had to be chosen for the introduction of new genetic material to the patient’s afflicted cells. Though the candidates were many, an rAAV delivery system is the best choice.

AAVs are small non-enveloped viruses (genus Dependoparvovirus) with a single-strand DNA genome of 4.8kbp length flanked by two inverted terminal repeats (ITR) sequences.

Wild type AAVs can only be infused with a sequence of limited length and, for their production and replication, usually require the presence of another virus (e.g., Adenovirus).

A recombinant AAV system, however, solves this problem by producing the virus in cells transfected with a combination of three plasmids: one for capsid and replication proteins (pRepCap), one for transcriptional and replication-boosting factors (pHelper), and lastly a plasmid containing a full cassette designed for protein expression of choice flanked by two ITRs. This way, not only can the 4.8kbp capacity of the virus be solely devoted to the cassette of choice, but the resulting viral particles are incapable of replication and viral protein expression, making it the ideal model for clinical applications. The serotype we have chosen is AAV2. Namely, the advantages of an rAAV2 delivery system are the following:

Safety: It is completely safe for the patient due to its very low immunogenicity and cytotoxic effects, since the only response of the body is the production of capsid-neutralizing antibodies. As of now, AAVs are the “stealthiest” viruses used in commercial gene therapies.

No Tumorigenicity: The viral DNA enters the nucleus and forms an episome, a circular DNA molecule separate from the chromosomal DNA. Even when the viral DNA is embedded in the genome, it is inserted in a known site in Chromosome 19, which does not have any tumorigenic effects. This would be a big problem if the virus we used was a retrovirus because they behave unexpectedly and embed themselves in random sites in the genomic DNA.

Long-term Stable Expression: of interest is contained in a very stable episome, which however loses its integrity with each replication. But this problem is not relevant in non-dividing cell lines, such as neurons, which we aim to target with our gene therapy model.

AAV2's Suitability: AAV2 is the most well-suited serotype for the job. In comparison to other serotypes, it boasts all of the above qualities and, at the same time, provides even higher transduction capabilities in certain tissues (tropism), especially towards the CNS and muscle, making it ideal for targeting neurons in Alzheimer's patients. Additionally, it is considered a high-yield serotype, making its production more efficient. Also, it is the most researched and well-known of the serotypes, making it the safest option.

Exosome Export: rAAVs can be exported in exosomes. The small capsid size of rAAVs allows it to enter exosomes. The viral particles inside exosomes have many times the transduction power of the intracellular ones.

Our project aims to harness the advantages of AAV-based gene therapy to deliver microRNA-195 to the neurons of Alzheimer's disease patients. To generate the recombinant AAV necessary for this purpose, three plasmids are required:

  1. pHelper Plasmid: Contains genes E2A, E4, and VA, essential for providing viral functions that AAVs cannot produce autonomously.
  2. Rep/Cap Plasmid: Encodes the genes required for AAV replication and capsid formation, facilitating functional viral particle production.
  3. Transfer Plasmid(pAAV-EF1a-mir195-eGFP-SV40pA): Contains our gene of interest, specifically designed to include microRNA-195.

Our rAAV vector cassette is carefully designed to include the DNA sequence for microRNA-195. Upon transduction, the rAAV genome forms an episome within the nucleus of target cells, ensuring stable expression without integrating into the host cell's chromosomal DNA, minimizing the risk of unintended mutations.

Drug Delivery Systems: Exosomes and Other Nanoparticles

Polymeric nanoparticles and liposomes are widely used in drug delivery systems for various health conditions. However, liposomes can have immunogenicity, short circulation times, stability issues, and toxicity concerns. Polymeric nanoparticles raise similar biocompatibility and long-term safety concerns. On the other hand, exosomes are emerging as effective tools for drug delivery systems.

Exosomes are extracellular vesicles produced by all normal cells, responsible for intercellular communication and eliminating unnecessary substances, maintaining intracellular homeostasis. They offer advantages that make them suitable as drug carriers for Alzheimer's treatment, such as the ability to transport proteins, mRNAs, miRNAs, and lipids, low toxicity, good biocompatibility, and lack of immune response. Their small size allows them to penetrate deeper into tissues and the blood-brain barrier, targeting nerve cells effectively. Additionally, exosomes can be surface and genetically modified using Synthetic Biology methods.

exosomes

The Vexosome; derived from the words “viral” and “exosome”, this molecular synergy is what drives the novelty of our gene therapy model. With this combination, the genome altering power of an rAAV viral vector can be transported through blood and tissue to the target cells shielded and concealed inside the body's own communication vehicles. This solves one of the main problems of virus based gene therapy, which is the immune reaction the virus causes in the recipient's body. An immune reaction not only is a very unwanted side effect, especially in AD patients, but an immune reaction also lowers the efficiency of the gene therapy since it attacks and degrades viral particles. When, however, the viral particles are carried inside exosomes, this phenomenon is completely avoided, providing a safe yet effective gateway to gene therapy delivery.

iPSCs: The cell line that will provide the “exosome” in our “vexosomes” was carefully chosen. For Project Lethe, iPSCs are greatly important, for they are the cells that are meant to grow, be transfected with an rAAV plasmid combination and produce viral particles, which will escape with the exosomes the cells secrete. The easily acquirable nature, fast growing capabilities, and transfection availability of iPSCs outside the body, offers us an efficient way of producing a satisfactory amount of vexosomes. On a side note, since iPSCs are stem cells, even their exosomes without viral particles have been shown to possess rejuvenating qualities, promoting cell health repair and maintenance, adding to the therapeutic profile of our approach.

Solution



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  1. How it all started?
  2. Why Lethe?
  3. The problem
  4. Symptomatology and Stages
  5. Pharmaceutical Approach
  6. Solution