Part:BBa_K5062050
CAR_Ma Cas12a Multiplex
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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 multiplexed CRISPR knockout with Cas12a (BBa_K5062021) and a guide RNA (gRNA) release system (BBa_K5062045)
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 multiplexed CRISPR/Cas12a-Mediated Knockout and guide RNA (gRNA) release system
Cas12a performs a similar function to Cas9 endonuclease, creating a double-strand break within the cell genome at a location with a complementary sequence to a guide RNA. However, unlike Cas9 which requires a crRNA and tracrRNA, Cas12a only uses short mature crRNAs (Zetsche et al., 2015), allowing for a more compact design. Furthermore, Cas12a can use arrays encoding four distinct gRNA sequences (Anvar et al., 2024), as opposed to Cas9 with only one gRNA sequence. The four gRNA sequences used with Cas12a are complementary to the SIRPa, PD-1, VTCN1 and STAB1 domains, resulting in the knock-out of the four genes within the cell genome. The Cas12a system can be used to target various genes depending on the array of gRNAs used, thus allowing for high degrees of flexibility and customization.
A diagram depics the differnces between Cas9 and Cas12a (Froehlich, J. J., 2024)
This also includes a release system designed for the release of anti-SIRPa guide RNA (gRNA) (BBa_K5062022), anti-PD1 gRNA (BBa_K5062024), anti-VTCN1 gRNA (BBa_K5062026) and STAB1 gRNA (BBa_K5062028) to allow for multiplexed Cas12a-mediated CRISPR operations. 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 shRNA 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 mulitple base-perfect shRNA.
The Cas12a mechanism works similarly to Cas9, with the difference of four separate guide RNAs composed of only the crRNA, thus being able to knock out up to 4 targets, rather than just 1 target with Cas9 (Anvar et al., 2024). In our design, aside from the SIRPα domain which is a crucial immune evasion checkpoint (Ray et al., 2018), three other targets have been chosen to increase the efficacy of our CAR macrophage. The PD1-PDL1 pathway, which acts as a T-cell activation checkpoint, and immune evasion checkpoint (Feng et al., 2019). STAB1, which involves inhibition of phagocytosis (Hollmén et al., 2020). As well as the VTCN1 which plays a role in T-cell inhibition (Podojil & Miller, 2017). Targeting these domains allows our macrophage to increase phagocytic ability and boost T-cell activity, overall targeting immune evasion with an increased efficacy.
A diagram depics the differnces between Cas9 and Cas12a (Froehlich, J. J., 2024)
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
Anvar, N. E., Lin, C., Ma, X., Wilson, L. L., Steger, R., Sangree, A. K., Colic, M., Wang, S. H., Doench, J. G., & Hart, T. (2024). Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform. Nature Communications, 15(1). https://doi.org/10.1038/s41467-024-47795-3
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
Feng, M., Jiang, W., Kim, B. Y. S., Zhang, C. C., Fu, Y., & Weissman, I. L. (2019). Phagocytosis checkpoints as new targets for cancer immunotherapy. Nature Reviews. Cancer, 19(10), 568–586. https://doi.org/10.1038/s41568-019-0183-z
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
Froehlich, J. J. (2024, August 18). Cas12a and Cas9 nucleases and their DNA cleavage positions. https://en.wikipedia.org/wiki/Cas12a#/media/File:Cas12a_vs_Cas9_cleavage_position.svg
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
Hollmén, M., Figueiredo, C. R., & Jalkanen, S. (2020). New tools to prevent cancer growth and spread: a ‘Clever’ approach. British Journal of Cancer, 123(4), 501–509. https://doi.org/10.1038/s41416-020-0953-0
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
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
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
Podojil, J. R., & Miller, S. D. (2017). Potential targeting of B7‐H4 for the treatment of cancer. Immunological Reviews, 276(1), 40–51. https://doi.org/10.1111/imr.12530
Ray, M., Lee, Y., Hardie, J., Mout, R., Tonga, G. Y., Farkas, M. E., & Rotello, V. M. (2018). CRISPRed macrophages for Cell-Based Cancer immunotherapy. Bioconjugate Chemistry, 29(2), 445–450. https://doi.org/10.1021/acs.bioconjchem.7b00768
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
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
Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., Van Der Oost, J., Regev, A., Koonin, E. V., & Zhang, F. (2015). CPF1 is a single RNA-Guided endonuclease of a Class 2 CRISPR-CAS system. Cell, 163(3), 759–771. https://doi.org/10.1016/j.cell.2015.09.038
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