Blog Post

Immune Cells: A “Natural” Path to Curing Disease

**This Blog post has not been Peer Reviewed**

The immune system is a versatile network of cells and cellular processes that is designed to prevent disease, whether it originates from foreign entities such as viruses or from internal sources such as cancer. Efforts towards improving the immune response for specific occurrence of disease are being increasingly explored. Taking advantage of the functionality of the immune system is an alluring therapeutic strategy that reduces the challenges associated with the use of exogenous drugs or chemotherapeutics. In this blog, we will discuss the basic understanding of the involvement of immune system in disease fighting and the current engineering tools being used to enhance the ability of immune system as a therapeutic strategy.

The Immune System and Its Role in Disease

There are two classes of immunity, namely inherited and acquired. Inherited refers to immunity gained through genetic inheritance while acquired refers to immunity developed by exposure to foreign entities. The immune system, whether inherited or required, responds with the activation of immune cells. Immune cells, also known as white blood cells, are cell populations that are specifically designed to detect, respond, and eradicate any entity that is considered dangerous to the body. Such immune cells include, but are not limited to, lymphocytes (T-cells and B-cells), natural killer cells and macrophages [1].

White blood cells are designed to recognize Damage-Associated Molecular Patterns (DAMPS) and respond to them, typically by eradicating the source, which could be a pathogenic cell [2]. The immune response is essential for maintaining the overall health of the cellular environment but chronic or more malevolent diseases such as cancer can avoid the immune system via evasion and/or suppression [2]. For example, mutations of the Protein death ligand-1 (PD-L1) gene, which is a significant marker for immune cell response, can cause a decrease in response, due to a loss of expression of PD-L1.. The change of expression in this case can allow the cancer cells, commonly in leukemia, to evade cell death and therefore propagate unchecked [2]. Immune cell evasion coupled with the unique tumor microenvironment [3], could make it difficult for a strong immune response to occur.

This is where the immunotherapies and engineering of immune cells comes into the picture. The tailored immune cells take over the responsibility to remove the disease cells at an effective rate without the need of toxic materials such as chemotherapies. To use immune cells for more long-term malignancies, engineering can be done to improve the functionality and targeting capacity of immune cells such that immune evasion cannot occur [4]. Furthermore, as genetic diseases such as cancer can have a multitude of different types of mutations between patients, the use of immune cells can provide a more tailored treatment option as they can be engineered on a case-by-case basis.

Tools of Immune Cell Engineering: Nucleic acid Technologies

Effective genetic modification is the key to produce immune cells for therapeutic use. This can occur either by modification of gene expression, modification of the gene itself (genetic editing), or through introduction of new genes for expression. There are several nucleic acid-based technologies being explored for developing immunotherapies:

1) Expression of recombinant genes by successful genome integration

Current genome integration in immunotherapy development is undertaken by using viral vectors. However, as these strategies can cause undesired features in modified cells due to viral vectors, alternative methods of delivery are being explored. One such method is the use of DNA transposons, which are genetic elements that can integrate into different regions of the genome [5]. DNA transposons consist of the transposase gene flanked by inverted terminal repeats (ITRs) that are recognized by the transposase. Once present, the transposase cuts at the ITR sites and, upon insertion, the ITR sites turn into target site duplications (TSD). Transposons typically insert into non-essential regions of the genome, preventing any possible loss of function of essential genes. As a tool in genetic editing, the gene that is to be inserted is flanked with the TIR sites and the transposase is preferably expressed (or introduced) separately, allowing for integration of the new gene [6]. The transposon DNA with TIR flanks and the transposase gene can be delivered via plasmid DNAs (pDNA) or as mRNA which can be applied to the immune cells ex vivo or in situ [7]. The use of transposons for CAR T-cell development has been explored as a means to treat acute leukemias [8].

2) Knockout, mutation, or insertion of genes via CRISPR/Cas9 technology

CRISPR (clustered regularly interspaced short palindromic repeats) is a technology that can effectively target and edit regions of the genome. Gene editing can occur by knockout or point mutations via Non-Homologous End-Joining (NHEJ) or by insertion of new genes via Homologous Recombination (HR) [9]. Compared to its predecessors such as TALENS (Transcription activator-like effector nucleases) and ZFNs (zinc-finger nucleases ) [10], CRISPR/Cas technology is a more promising gene editing tool due to its ability to target specific regions of the genome. Specific RNA sequences that are homologous to the target sequence (typically 20 nucleotides) are fused to an RNA scaffold. This scaffold aids the delivery of the Cas endonuclease which then cleaves at the target site. With the edition of “donor DNA” (DNA that is to be inserted into the genome that contains homologous overhangs to both sides of the cleavage site), new sequences can then be integrated into the genome [11]. The highly specific nature of gene editing makes this technology a desirable tool for tailoring the right cells for immunotherapy.

3) Regulation of gene expression with the use of siRNA, shRNA and miRNA

RNA interference (RNAi) is a popular method for genetic modulation as it can only induce transient knockdown of genes as opposed to permanent knockout. RNAi can occur through the use of either siRNA (short interfering RNA) that is supplied exogenously, or shRNA (short hairpin RNA) that is either expressed via a plasmid DNA or after genomic integration [12]. Both approaches essentially tap into the inherent RNAi mechanism, but differ in terms of pre-processing within the cell [13]. Once active, the siRNA or shRNA find the target transcripts and cause degradation of the mRNA, decreasing the amount of protein translation. The use of RNAi technology has resulted in FDA approved drugs and is expected to show success in clinical trials of immunotherapy [14]. The microRNA (miRNA) approach is another regulator of gene expression. Such short RNAs (20-24 nucleotides) can repress protein translation by binding to messenger RNA (mRNA) and increasing the rate of natural mRNA decay. Some miRNAs have tumor suppressor or oncogenic activities within cancer and other diseases, and are therefore transcribed at lower or higher levels in some cancers respectively [15]. microRNA can act as a driver of specific cancers, but due to their repression of protein translation, can also act as a genetic modification tool. Inhibition or induction of miRNA expression, depending on its role within the immune cell, can improve the immune response to different cancer types [16].

Tools of Immune Cell Engineering: Delivery Vehicles

Despite the power of nucleic acid driven genetic modifications, the low stability of the nucleic acids and the difficulty of nucleic acid transport across cell membranes make the implementation of this therapeutic strategy challenging. A delivery vehicle is required for successful internalization of nucleic acids; a shift from the current ‘norm’ of viral vectors in immunotherapies towards non-viral delivery methods is attractive as it causes less cytotoxic effects. A plethora of non-viral delivery methods being explored, including the RJH Biosciences’ lipopolymers, are leading to effective replacements. The current bottleneck for these technologies is their characterization and use beyond the pre-clinical research. Safer and more reliable technologies are bound to emerge as more delivery vehicles are moved into the clinical testing phase. As one of the crucial steps of immunotherapies is quick integration of nucleic acids for genetic modification, the constant improvement of delivery vehicles is required for more successful therapeutics.

The use of lipopolymers in the clinical field is one of the main goals of RJH Biosciences. Our products are incredibly effective for modification of suspension cells and therefore are advantageous for immunotherapy development. For partnership opportunities or for more information on our clinical goals, please email us at rjhbiosciences@gmail.com. RJH Biosciences is a company dedicated to developing and commercializing the best transfection reagents or delivery vehicles possible.

References:

  1. Haseeb, M.; Anwar, M.A.; Choi, S. Molecular Interactions Between Innate and Adaptive Immune Cells in Chronic Lymphocytic Leukemia and Their Therapeutic Implications. Front Immunol 2018, 9, 2720, doi:10.3389/fimmu.2018.02720.
  2. Curran, E.K.; Godfrey, J.; Kline, J. Mechanisms of Immune Tolerance in Leukemia and Lymphoma. Trends Immunol 2017, 38, 513-525, doi:10.1016/j.it.2017.04.004.
  3. Loew, A.; Kohnke, T.; Rehbeil, E.; Pietzner, A.; Weylandt, K.H. A Role for Lipid Mediators in Acute Myeloid Leukemia. Int J Mol Sci 2019, 20, doi:10.3390/ijms20102425.
  4. Lim, W.A.; June, C.H. The Principles of Engineering Immune Cells to Treat Cancer. Cell 2017, 168, 724-740, doi:10.1016/j.cell.2017.01.016.
  5. Kebriaei, P.; Izsvak, Z.; Narayanavari, S.A.; Singh, H.; Ivics, Z. Gene Therapy with the Sleeping Beauty Transposon System. Trends Genet 2017, 33, 852-870, doi:10.1016/j.tig.2017.08.008.
  6. Munoz-Lopez, M.; Garcia-Perez, J.L. DNA transposons: nature and applications in genomics. Curr Genomics 2010, 11, 115-128, doi:10.2174/138920210790886871.
  7. Tipanee, J.; Chai, Y.C.; VandenDriessche, T.; Chuah, M.K. Preclinical and clinical advances in transposon-based gene therapy. Biosci Rep 2017, 37, doi:10.1042/BSR20160614.
  8. Magnani, C.F.; Tettamanti, S.; Alberti, G.; Pisani, I.; Biondi, A.; Serafini, M.; Gaipa, G. Transposon-Based CAR T Cells in Acute Leukemias: Where are We Going? Cells 2020, 9, doi:10.3390/cells9061337.
  9. Antony, J., Haque, A., Lamsfus-Calle, A., Daniel-Moreno, A., Mezger, M., and Kormann, M. CRISPR/Cas9 system: A promising technology for the treatment of inherited and neoplastic hematological diseases. Advances in cell and gene therapy 2018, 1, e10.
  10. Gaj, T.; Sirk, S.J.; Shui, S.L.; Liu, J. Genome-Editing Technologies: Principles and Applications. Cold Spring Harb Perspect Biol 2016, 8, doi:10.1101/cshperspect.a023754.
  11. Doudna, J.A.; Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014, 346, 1258096, doi:10.1126/science.1258096.
  12. Uludag, H.; Ubeda, A.; Ansari, A. At the Intersection of Biomaterials and Gene Therapy: Progress in Non-viral Delivery of Nucleic Acids. Front Bioeng Biotechnol 2019, 7, 131, doi:10.3389/fbioe.2019.00131.
  13. Rao, D.D.; Vorhies, J.S.; Senzer, N.; Nemunaitis, J. siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev 2009, 61, 746-759, doi:10.1016/j.addr.2009.04.004.
  14. Ramishetti, S.; Peer, D. Engineering lymphocytes with RNAi. Adv Drug Deliv Rev 2019, 141, 55-66, doi:10.1016/j.addr.2018.12.002.
  15. Wallace, J.A.; O'Connell, R.M. MicroRNAs and acute myeloid leukemia: therapeutic implications and emerging concepts. Blood 2017, 130, 1290-1301, doi:10.1182/blood-2016-10-697698.
  16. Cortez, M.A.; Anfossi, S.; Ramapriyan, R.; Menon, H.; Atalar, S.C.; Aliru, M.; Welsh, J.; Calin, G.A. Role of miRNAs in immune responses and immunotherapy in cancer. Genes Chromosomes Cancer 2019, 58, 244-253, doi:10.1002/gcc.22725.

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