Part:BBa_K5062048

CAR_Ma Mutliplexed shRNA

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2092
  Illegal EcoRI site found at 10240
  Illegal SpeI site found at 2040
  Illegal SpeI site found at 8557
  Illegal PstI site found at 4413
  Illegal PstI site found at 7716
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2092
  Illegal EcoRI site found at 10240
  Illegal NheI site found at 5284
  Illegal NheI site found at 6393
  Illegal NheI site found at 7210
  Illegal SpeI site found at 2040
  Illegal SpeI site found at 8557
  Illegal PstI site found at 4413
  Illegal PstI site found at 7716
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2092
  Illegal EcoRI site found at 10240
  Illegal BglII site found at 2214
  Illegal BamHI site found at 2076
  Illegal BamHI site found at 2439
  Illegal BamHI site found at 7347
  Illegal XhoI site found at 5448
  Illegal XhoI site found at 5509
  Illegal XhoI site found at 5550
  Illegal XhoI site found at 12326
  Illegal XhoI site found at 12441
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2092
  Illegal EcoRI site found at 10240
  Illegal SpeI site found at 2040
  Illegal SpeI site found at 8557
  Illegal PstI site found at 4413
  Illegal PstI site found at 7716
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2092
  Illegal EcoRI site found at 10240
  Illegal SpeI site found at 2040
  Illegal SpeI site found at 8557
  Illegal PstI site found at 4413
  Illegal PstI site found at 7716
  Illegal NgoMIV site found at 1603
  Illegal NgoMIV site found at 2881
  Illegal AgeI site found at 4989
  Illegal AgeI site found at 5358
  Illegal AgeI site found at 7353
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 975
  Illegal BsaI site found at 5547
  Illegal BsaI site found at 5565
  Illegal BsaI site found at 8312
  Illegal BsaI.rc site found at 2344
  Illegal BsaI.rc site found at 2991
  Illegal BsaI.rc site found at 5074
  Illegal BsaI.rc site found at 7817
  Illegal BsaI.rc site found at 9524
  Illegal BsaI.rc site found at 12118
  Illegal SapI.rc site found at 9729
  

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)
• Multiplexed RNA Interference via a dual short Hairpin RNA (shRNA) release system(BBa_K5062016)

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.

In vitro characterisation of this part is currently ongoing prior to the wiki freeze. Full part characterisation is expected by the 2024 iGEM Giant Jambouree.

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

Multiplexed Short Hairpin RNA (shRNA) Release System

Short Hairpin RNA (shRNA) is indeed a single-stranded RNA that folds back on itself forming a stem-loop structure. This loop is what gives the shRNA the appearance of a double-stranded RNA (dsRNA). The non-complementary region of the RNA sequence forms the loop that connects the two ends of the stem, allowing it to fold back on itself.

Once the shRNA is introduced into the host cell, the shRNA is transcribed by its promoter. The transcribed shRNA forms a stem-loop structure which is then transported from the nucleus to the cytoplasm by the action of Exportin 5. Then it is processed by an endoribonuclease known as “DICER” which cleaves the loop and converts the shRNA into short dsRNAs or small interfering RNA (siRNA).

Upon getting processed by DICER, the dsRNA is then loaded into the RNA-induced silencing complex (RISC), where the sense (passenger) strand gets degraded, leaving the antisense (guide) strand to guide the RISC complex towards the complementary target mRNA sequence (O’Keefe, 2013). A crucial component of RISC that plays a central role in the gene silencing mechanism is the presence of Argonaute 2 (Ago2) protein in RISC. Ago2 possesses endonuclease activity which allows it to cleave the target mRNA at the target site when the guide strand perfectly complements the target strand (Ruda et al., 2014). This cleavage results in the degradation of the target mRNA, leading to effective silencing of the target gene.

In our case, the guide strand of the designed shRNA binds to the mRNA of SIRPα leading to its cleavage and degradation by RISC. This cleavage of SIRPα results in a significant reduction in the expression levels of SIRPα which enhances the phagocytic activity. As the blockage of the SIRPa/CD47 pathway leads to a 4-fold increase in phagocytotic ability (Ray et al., 2018), the blockage of PD1/PDL1 also leads to a significant increase in phatocytotic ability (Feng et al., 2019). With the simultaneous disrupttion of both SIRPa/CD47 and PD1/PDL1 has an even greater effect on improving phagocytotic ability and cytokine release, boosting the immune system response against tumors (Lian et al., 2019).

A simplified diagram depicting the processing of shRNA in vivo leading to transcriptional regulation (Fouquerel et al., 2014)

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

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

Fouquerel, E., Li, J., Braganza, A., Yu, Z., Brown, A. R., Wang, X., Schamus, S., Svilar, D., Fang, Q., & Sobol, R. W. (2014). Use of RNA interference to study DNA repair. In Methods in pharmacology and toxicology (pp. 413–447). https://doi.org/10.1007/978-1-4939-1068-7_24

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

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

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

O’Keefe, E. P. (2022, October 29). SiRNAs and SHRNAs: Tools for protein knockdown by gene Silencing. https://www.labome.com/method/siRNAs-and-shRNAs-Tools-for-Protein-Knockdown-by-Gene-Silencing.html

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

Ruda, V. M., Chandwani, R., Sehgal, A., Bogorad, R. L., Akinc, A., Charisse, K., Tarakhovsky, A., Novobrantseva, T. I., & Koteliansky, V. (2014). The roles of individual mammalian argonautes in RNA interference in vivo. PLoS ONE, 9(7), e101749. https://doi.org/10.1371/journal.pone.0101749

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

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

Wu, L., Wei, Q., Brzostek, J., & Gascoigne, N. R. J. (2020). Signaling from T cell receptors (TCR