Team:MIPT MSU/Implementation

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Implementation

Proposed Implementation

Overview

Developing our project we aimed on creating a new way of targeted drug delivery, based on vesicles with syncytin. Our construction allows delivery of drugs and other biologically active molecules to colorectal and breast cancer through synthetic receptors on its cells. Moreover, we believe that our system might find application in other area.

Proposed end Users

The prototype of our system is proposed for patients with some cancer types with overexpression of the receptors for syncytin (including breast, colorectal and so forth) [1]. Using psi signals (signals of packaging) the RNA sequence of interest can be packed into a vesicle via gag protein [2]. Different molecules can be loaded into the vesicle through the mechanism by which the psi signal adds to almost any sequence using simple cloning pipelines. This flexibility allows therapists to personalize the desired effect. For example, the RNA sequence can encode the non-mutated TP53 sequence or gRNA for knock out of mutated/overexpressed genes.

Moreover, our product boasts even bigger flexibility - by replacing the syncytin with another fusion protein with a different receptor we can acquire an opportunity to deliver our drug or RNA to another tissue. These advantages can be used to develop methods for therapy of other cancers or maybe even some tissue specific single-gene disorders.

The proposed variants of fusion proteins you can see in a table below made by our PI and us [6]:

Advantages over previous target delivery systems

Our system has some remarkable advantages over other target delivery systems such as nano-particles and viruses. First of them is decreased immunogenicity. Since vesicles are integral parts of our organisms they will not cause some heavy immune response and thus are much more safe than viruses and nanoparticles. Also our system boasts high tissue specificity because of fusion protein - receptor protein interaction specificity. Many nanoparticles do not have such properties. [3] In addition, it is very important that due to proteins, the fusion of the vesicles' contents bypasses degradation in the endosomal pathway.

Proposed factoring

Before usage in clinical practice the large amount of the vesicles should be produced and purified

We think this pipeline can represent the factoring of our product:

  1. Growing the vesicle producing cell line in serum-free medium.
  2. Collecting the medium with vesicles.
  3. Purifying vesicles on a column (300 kDa size).
  4. Additional purifying stages. (up to decide).
  5. Quality control using lipophilic dye (PKH 26).
  6. Cooled vesicles can be stored in a kelvinator (-80 C) but the fine condition of storing is still up to decide.

Our vision

We see our project as an effective cancer therapy, which can with minimal risks decrease cancer progression and eliminate tumors. The drug will be delivered to a tumor through blood with high specificity.

Challenges to be Met

  1. Before use, the purified vesicles on the column must undergo other purification steps that will be developed.
  2. We should take it into consideration that the vesicle producing cell line contains its own HLA, which can cause inflammation and other unpleasant reactions if it appears on the vesicle surface. To avoid this we came up with the two following ideas:
    1. Genetically edit the cell line to delete HLA [4];
    2. Use cells of patient to create primary cell line.

    Both of these approaches can be tested.

  3. The storage of vesicles is also a complicated question. [5]
  4. The route of drug delivery to the tumor should be considered according to experiments and modeling as well.

Safety

Our team is very concerned about safety. To make our product safe we proposed a several decisions, including:

  1. Use non cancer cell lines to produce vesicles to avoid unexpected aftereffects.
  2. Use a serum-free medium to avoid collecting its proteins and miRNA with vesicles.
  3. Make sure that our project will not cause an immune response via HLA like it described above.
  4. Conduct additional purification stages.
  5. Make sure that our vesicles are sterile.

References

  1. Olson ED, Musier-Forsyth K. Retroviral Gag protein-RNA interactions: Implications for specific genomic RNA packaging and virion assembly. Semin Cell Dev Biol. 2019 Feb;86:129-139. doi: 10.1016/j.semcdb.2018.03.015. Epub 2018 Apr 1. PMID: 29580971; PMCID: PMC6167211.
  2. Larsen JM, Christensen IJ, Nielsen HJ, Hansen U, Bjerregaard B, Talts JF, Larsson LI. Syncytin immunoreactivity in colorectal cancer: potential prognostic impact. Cancer Lett. 2009 Jul 18;280(1):44-9. doi: 10.1016/j.canlet.2009.02.008. Epub 2009 Mar 26. PMID: 19327884.
  3. Ronzitti G, Gross DA, Mingozzi F. Human Immune Responses to Adeno-Associated Virus (AAV) Vectors. Front Immunol. 2020;11:670. Published 2020 Apr 17. doi:10.3389/fimmu.2020.00670.
  4. Amiri, F., Ranjbar, M., Pirouzfar, M. et al. HLA-A gene knockout using CRISPR/Cas9 system toward overcoming transplantation concerns. Egypt J Med Hum Genet 22, 37 (2021). https://doi.org/10.1186/s43042-021-00155-y.
  5. Jeyaram A, Jay SM. Preservation and Storage Stability of Extracellular Vesicles for Therapeutic Applications. AAPS J. 2017;20(1):1. Published 2017 Nov 27. doi:10.1208/s12248-017-0160-y.
  6. Zubarev, I.; Vladimirtsev, D.; Vorontsova, M.; Blatov, I.; Shevchenko, K.; Zvereva, S.; Lunev, E.A.; Faizuloev, E.; Barlev, N. Viral Membrane Fusion Proteins and RNA Sorting Mechanisms for the Molecular Delivery by Exosomes. Preprints 2021, 2021100130 (doi: 10.20944/preprints202110.0130.v1).