Composite

Part:BBa_K5062049

Designed by: Christian Nelson Tejedor Vicera   Group: iGEM24_HKU-HongKong   (2024-09-28)


CAR_Ma Cas9

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2293
    Illegal EcoRI site found at 10441
    Illegal SpeI site found at 2241
    Illegal SpeI site found at 8758
    Illegal PstI site found at 4614
    Illegal PstI site found at 7917
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2293
    Illegal EcoRI site found at 10441
    Illegal NheI site found at 5485
    Illegal NheI site found at 6594
    Illegal NheI site found at 7411
    Illegal SpeI site found at 2241
    Illegal SpeI site found at 8758
    Illegal PstI site found at 4614
    Illegal PstI site found at 7917
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2293
    Illegal EcoRI site found at 10441
    Illegal BglII site found at 2415
    Illegal BamHI site found at 2277
    Illegal BamHI site found at 2640
    Illegal BamHI site found at 7548
    Illegal XhoI site found at 5649
    Illegal XhoI site found at 5710
    Illegal XhoI site found at 5751
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2293
    Illegal EcoRI site found at 10441
    Illegal SpeI site found at 2241
    Illegal SpeI site found at 8758
    Illegal PstI site found at 4614
    Illegal PstI site found at 7917
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2293
    Illegal EcoRI site found at 10441
    Illegal SpeI site found at 2241
    Illegal SpeI site found at 8758
    Illegal PstI site found at 4614
    Illegal PstI site found at 7917
    Illegal NgoMIV site found at 1804
    Illegal NgoMIV site found at 3082
    Illegal AgeI site found at 5190
    Illegal AgeI site found at 5559
    Illegal AgeI site found at 7554
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1176
    Illegal BsaI site found at 5748
    Illegal BsaI site found at 5766
    Illegal BsaI site found at 8513
    Illegal BsaI.rc site found at 2545
    Illegal BsaI.rc site found at 3192
    Illegal BsaI.rc site found at 5275
    Illegal BsaI.rc site found at 8018
    Illegal BsaI.rc site found at 9725
    Illegal BsaI.rc site found at 12319
    Illegal SapI.rc site found at 9930

Overview

This is CAR_Ma, a multi-modal RNA powering a next-generation immunotherapy.

This is the combination of:

  • Transient, sustained and controlled self-replicating RNA vector (BBa_K5062046, BBa_K4696003)
  • 5th Generation Nanobody-powered Chimeric Antigen Receptor (BBa_K5062007)
  • Transient CRISPR knockout with Cas9-GFP (BBa_K5062030) and a guide RNA (gRNA) release system (BBa_K5062039)

This part encodes the entire CAR_Ma multi-modal RNA. To use, place beween a strong promoter and terminator on a vector used for in-vitro transcription.

Due to a lack of time, this part will not be fully characterized by the 2024 Giant Jamboree, however it will be continually worked on post iGEM 2024.


Usage and Biology

Self-Replicating RNA

In Alphaviruses such as VEE, nsP1 is typically in charge of membrane-binding, anchoring and mRNA capping. nsP2 is a multi-functional protein with proteolytic, helicase and NTPase capabilities. nsP3 is a macrodomain and is also in charge of anchoring the other 3 nsPs together. While nsP4 is the RNA-dependant RNA polymerase (Bakar & Ng, 2018). During viral replications the nsPs are often expressed together as a polyprotein and cleaved by nsP2 into four separate proteins during maturation. Typically, we see nsP123 and nsP4 expressed directly or generated by cleaving the nsP1234 early on.

A diagram depics the schamatic of Alphavirus open reading frames (Bakar & Ng, 2018)

This nsP1 sequence was derived from the Venezuelan equine encephalitis (VEE) virus, and has a role in replicating genomic or subgenomic viral RNA (Kim & Diamond, 2022). A complete nsP1-4 protein forms a replication complex, which will bind to the 26 subgenomic promoter to initiate downstream gene expression (Mc Cafferty et al., 2021). This part is useful in the construction of self-replicating RNA-based constructs when cloned alongside nsp2, 3, and 4. It should be cloned upstream of nsP2, and upstream of any subgenomic promoter, such as 26S/-26S. nsP1, 2, and 3 should form an uninterrupted sequence in the construct for appropriate expression of the replicase complex.

Further, built-on to the self-replicating RNA system is a aariable control switch modulated by trimethoprim (TMP). A highlight of the multi-modal RNA system allowing us to regulate both full-system replication and subgenomic expression. By manuiplating the dose of TMP in the system, we are able to vary the expression of proteins such as the chimeric antigen receptor, but also modulate the release of short hairpin (RNA) to adjust the strength of transcriptomic silencing and further regulate the replication of the entire system. This allows us to maximise efficiency of the system while minimising cellular burden from excess protein expression or RNA replication. In addition to this, this grants us the additional ability to quickly shutoff the full system in case of treatment completion or immunogenic emergencies, granting an extra level of safety.

The study by Cafferty, S. M. et al. (2020) depics the increase in expression as TMP decreases demonstrating the shut-off effect of this system.


5th Generation Nanobody-powered Chimeric Antigen Receptor

This sequence encodes an entire CAR polyprotein used for testing. Due to the complexity of this protein, it is unable to be cloned via the iGEM cloning standards. Rather, we recommend designing primers to clone this on to subsequent systems via NEBuilder as done by HKU-HongKong. Further, by using primers the RFP mCherry XL can also be removed allowing for removal if desired for use in downstream clinical applications.

This 5th Generation Chimeric Antigen Receptor has the following features:

  • An anti-GPC3 nanobody targeting domain enabling targeting of tumors such as hepatocellular carcinoma, squamous non-small cell lung cancer and breast cancer. Nanobodies hold advantages over traditional scFvs by being smaller, easier to engineer, and less immunogenic (Jin et al., 2023).
  • Optimized human co-stimulatory domains with CD19-FcγR Tandem and MegF10 inspired from the work of Morrissey, M. A. et al. (2018) and their work with mouse phagocytes. These co-stimulatory domains enhance phagocytosis, trogocytosis, engulfment of large targets, antigen presentation and macrophage micration.
  • Human IFNγ Armouring Domain allowing for the sustained M1 poloarization of macrophages enforcing their anti-tumor effect (Jorgovanovic et al., 2020). The co-expression of IFNγ also allows for the reprogramming of nearby tumor-associated macropahges (TAMs) within the tumor microenvironment (TME).
  • Variable control switch modulated by trimethoprim (TMP). A highlight of the 5th Generation CAR is the ability to control the strength of CAR expression by leveraging TMP-stabilized DD-L7Ae.

A schematic demonstrating the architechture of earlier generations of Chimeric Antigen Receptor (Wu et al., 2020).


The study by Morrissey, M. A. et al. (2018) depics the improvement in phagocytotic ability with co-stimulatory domains such as FcγR and MegF10


Transient CRISPR/Cas9-Mediated Knockout and guide RNA (gRNA) release system

Cas9 (Csn1) endonuclease originates from the Streptococcus pyogenes Type II CRISPR/Cas system (Xu et al., 2019), assembled with GFP from Aequorea victoria. Cas9 can be used with a guide RNA to cleave genomic DNA at a specified location and is a common tool in gene editing, while GFP is used as a fluorescent marker and can be used to track the localization and presence of labelled molecules (Chudakov & Lukyanov, 2003). Both Cas9 and GFP are placed within the same reading frame to assemble a Cas9-GFP fusion protein, which has been shown to increase the mutagenesis and efficiency of the Cas9 endonuclease, without a significant increase in off-target effects (Park et al., 2021).

The Cas9 mechanism involves the formation of a Cas9 complex, consisting of the Cas9 protein and a guide RNA (gRNA). The gRNA is composed of the target-specific crRNA, whose sequence is complementary to the target domain, and the scaffolding tracrRNA, which allows for binding with the Cas9 endonuclease. When formed, the gRNA is used to direct the Cas9 enzyme to a specific gene of interest, where the Cas9 enzyme is then able to induce a double-strand break at the specific site, being the SIRPα domain (Kim et al., 2014). With the use of cell’s natural DNA repair pathways, such as non-homologous end joining, the excised DNA is repaired, however, small insertions and deletions (indels) are introduced at the junction. As such, the error-prone DNA sequence results in disrupted protein expression (Ishibashi et al., 2020). The final product is a SIRPα-deficient macrophage, able to interrupt the SIRPα-CD47 pathway and thus preventing the immune evasion of tumor cells, while allowing for phagocytosis and an overall increase in the efficacy of CAR macrophages.


The action of Cas9 (Ding et al., 2016)


This also includes a release system designed for the release of Cas9 guide RNA (gRNA) (BBa K5062020) to allow for CRISPR to disrupt the expression of SIRPa. The system utilizes a 5' Hammerhead Ribozyme (BBa_K5062009) and a 3' Hepatitis Delta Virus (HDV)(BBa_K5062010) Ribozyme to catalytically self-cleave any unnecessary sequences at 5' and 3' ends of the shRNA allowing for its exact sequence to be preserved.

This system allows gRNA expression using any RNA polymerase, including those that inherently contain capping and tailing capabilities such as the RNA polymerase of Venezuelan equine encephalitis (VEE) virus which powers self-replicating RNA (srRNA) systems, allowing a transient srRNA system to sustain the subgenomic expression and release of base-perfect gRNA.

The Cas9 mechanism requires the formation of a Cas9 complex, consisting of the Cas9 protein and a guide RNA (gRNA). The gRNA is composed of the target-specific crRNA, whose sequence is complementary to the target domain, and the scaffolding tracrRNA, which allows for binding with the Cas9 endonuclease. When formed, the gRNA is used to direct the Cas9 enzyme to a specific gene of interest, where the Cas9 enzyme is then able to induce a double-strand break at the specific site, being the SIRPα domain (Kim et al., 2014). With the use of cell’s natural DNA repair pathways, such as non-homologous end joining, the excised DNA is repaired, however, small insertions and deletions (indels) are introduced at the junction. As such, the error-prone DNA sequence results in disrupted protein expression (Ishibashi et al., 2020). The final product is a SIRPα-deficient macrophage, able to interrupt the SIRPα-CD47 pathway and thus preventing the immune evasion of tumor cells, while allowing for phagocytosis and an overall increase in the efficacy of CAR macrophages.


A diagram depics the Cas9-gRNA interaction (The Complete Guide to Understanding CRISPR SGRNA, n.d.)


The self-cleavage reaction catalyzed by the Hammerhead Ribozyme involves a phosphodiester isomerization mechanism. This results in the formation of a 2',3'-cyclic phosphate at the 5' end and a 5'-hydroxyl group at the 3' end of the cleaved RNA. The reaction is reversible, allowing the Hammerhead Ribozyme to also catalyze the ligation of the cleaved RNA strands (Hammann et al., 2012).

The Hammerhead Ribozyme is considered one of the best-characterized ribozymes. Its small size, well-studied cleavage chemistry, known crystal structure, and biological relevance make it a useful model system for understanding the fundamental mechanisms of RNA catalysis. Hammerhead Ribozymes have been found ubiquitously across all domains of life, where they play roles in processes like rolling-circle replication of viral genomes and co-transcriptional processing of retrotransposons (Jimenez et al., 2015).

The sequence is only the core of the hammerhead ribozyme. A 6 nucleotide head should be added to the 5' end of the ribozyme core which is complementary to the first 6 nucleotides of the RNA to be released to allow a succesful cleavage. Since this is a DNA part, it should be added to the DNA that eventually transcirbes into the relevant RNA sequences.


A diagram depics the design considerations required for using the 5' Hammerhead Ribozyme (UC San Diego, n.d.)


The HDV ribozyme has a characteristic nested double-pseudoknot secondary structure with five helical regions (P1, P1.1, P2, P3, P4) (Ferré-D’Amaré & Scott, 2010). The HDV ribozyme catalyzes a site-specific transesterification reaction, where the 2' hydroxyl group of the nucleotide upstream of the cleavage site acts as a nucleophile to attack the adjacent phosphodiester bond. This results in the formation of a 2'-3' cyclic phosphate and a 5' hydroxyl product (Jimenez et al., 2015).

The HDV ribozyme can function through both metal-dependent and metal-independent mechanisms. In the metal-dependent mechanism, a divalent metal ion (typically Mg2+) helps to stabilize the developing negative charge on the 2' nucleophile and the pentavalent phosphorane transition state (Ke et al., 2004). In the metal-independent mechanism, the catalytic cytosine (C75) can act as a general acid to protonate the 5' leaving group (Ferré-D’Amaré & Scott, 2010).

The self-cleavage activity of the HDV ribozyme is essential for processing the HDV genomic and antigenomic RNA strands during rolling-circle replication (Ferré-D'Amaré, A.R et. al,2010). The cleavage generates linear RNA strands with 5' hydroxyl and 2'-3' cyclic phosphate ends, which can then be ligated to form the circular HDV genome (Jimenez, R.M. et al., 2015).

This can be added directly to the 3' end of RNA sequences. Since this is a DNA part, it should be added to the DNA that eventually transcirbes into the relevant RNA sequences.


A diagram depics the design considerations required for using the 3' HDV Ribozyme (UC San Diego, n.d.)


References

Bakar, F. A., & Ng, L. (2018). Nonstructural proteins of Alphavirus—Potential targets for drug development. Viruses, 10(2), 71. https://doi.org/10.3390/v10020071

Cafferty, S. M., De Temmerman, J., Kitada, T., Becraft, J. R., Weiss, R., Irvine, D. J., Devreese, M., De Baere, S., Combes, F., & Sanders, N. N. (2021). In vivo validation of a reversible small Molecule-Based switch for synthetic Self-Amplifying mRNA regulation. Molecular Therapy, 29(3), 1164–1173. https://doi.org/10.1016/j.ymthe.2020.11.010

Chudakov, D. M., & Lukyanov, K. A. (2003). Use of green fluorescent protein (GFP) and its homologs for in vivo protein motility studies. Biochemistry (Moscow), 68(9), 952–957. https://doi.org/10.1023/a:1026048109654

Ding, Y., Li, H., Chen, L., & Xie, K. (2016). Recent advances in genome editing using CRISPR/CAS9. Frontiers in Plant Science, 7. https://doi.org/10.3389/fpls.2016.00703

Ferré-D’Amaré, A. R., & Scott, W. G. (2010). Small self-cleaving ribozymes. Cold Spring Harbor Perspectives in Biology, 2(10), a003574. https://doi.org/10.1101/cshperspect.a003574

Hammann, C., Luptak, A., Perreault, J., & De La Peña, M. (2012). The ubiquitous hammerhead ribozyme. RNA, 18(5), 871–885. https://doi.org/10.1261/rna.031401.111

Jimenez, R. M., Polanco, J. A., & Lupták, A. (2015). Chemistry and Biology of Self-Cleaving Ribozymes. Trends in Biochemical Sciences, 40(11), 648–661. https://doi.org/10.1016/j.tibs.2015.09.001

Ishibashi, A., Saga, K., Hisatomi, Y., Li, Y., Kaneda, Y., & Nimura, K. (2020). A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-79303-0


Jin, B., Odongo, S., Radwanska, M., & Magez, S. (2023). Nanobodies: A Review of generation, Diagnostics and Therapeutics. International Journal of Molecular Sciences, 24(6), 5994. https://doi.org/10.3390/ijms24065994

Jorgovanovic, D., Song, M., Wang, L., & Zhang, Y. (2020). Roles of IFN-γ in tumor progression and regression: a review. Biomarker Research, 8(1). https://doi.org/10.1186/s40364-020-00228-x

Ke, A., Zhou, K., Ding, F., Cate, J. H. D., & Doudna, J. A. (2004). A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature, 429(6988), 201–205. https://doi.org/10.1038/nature02522

Kim, A. S., & Diamond, M. S. (2022). A molecular understanding of alphavirus entry and antibody protection. Nature Reviews Microbiology, 1–12. https://doi.org/10.1038/s41579-022-00825-7

Kim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J. (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research, 24(6), 1012–1019. https://doi.org/10.1101/gr.171322.113

Morrissey, M. A., Williamson, A. P., Steinbach, A. M., Roberts, E. W., Kern, N., Headley, M. B., & Vale, R. D. (2018). Chimeric antigen receptors that trigger phagocytosis. eLife, 7. https://doi.org/10.7554/elife.36688

Park, J., Yoon, J., Kwon, D., Han, M.-J., Choi, S., Park, S., Lee, J., Lee, K., Lee, J., Lee, S., Kang, K.-S., & Choe, S. (2021). Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins. Scientific Reports, 11(1), 16199. https://doi.org/10.1038/s41598-021-95406-8

Wagner, T. E., Becraft, J. R., Bodner, K., Teague, B., Zhang, X., Woo, A., Porter, E., Alburquerque, B., Dobosh, B., Andries, O., Sanders, N. N., Beal, J., Densmore, D., Kitada, T., & Weiss, R. (2018). Small-molecule-based regulation of RNA-delivered circuits in mammalian cells. Nature Chemical Biology, 14(11), 1043–1050. https://doi.org/10.1038/s41589-018-0146-9

The Complete Guide to Understanding CRISPR SGRNA. (n.d.). Synthego. https://www.synthego.com/guide/how-to-use-crispr/sgrna

UC San Diego. (n.d.). 3’ Ribozyme design. https://labs.biology.ucsd.edu/zhao/CRISPR_web/3_prime_ribozyme_design.html

UC San Diego. (n.d.). 5’ Ribozyme design. https://labs.biology.ucsd.edu/zhao/CRISPR_web/5_prime_ribozyme_design.html

Wu, L., Wei, Q., Brzostek, J., & Gascoigne, N. R. J. (2020). Signaling from T cell receptors (TCRs) and chimeric antigen receptors (CARs) on T cells. Cellular and Molecular Immunology, 17(6), 600–612. https://doi.org/10.1038/s41423-020-0470-3

Xu, X., Wan, T., Xin, H., Li, D., Pan, H., Wu, J., & Ping, Y. (2019). Delivery of CRISPR/Cas9 for therapeutic genome editing. The Journal of Gene Medicine, 21(7). https://doi.org/10.1002/jgm.3107


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