Engineering

Multi-Modal RNA

Given the inherent heterogeneity of cancer in its causation and progression, CAR_Ma was designed to be multi-modal in attempts to fully address the many facets of the complex disease, whilst at the same time optimizing the resource utilization and function of transfected macrophages against cancer cells. This year’s CAR_Ma was designed with elements that require three modal functions: transience, replicative, and release. The VEE replication complex designed into CAR_Ma facilitates the functioning of the different modalities, allowing for different sections of the RNA construct to be transient, repeated, or released.

Cancer is a Complex Problem Cancer in itself is unilaterally complex. A multifactorial disease capable of both sporadic and familial origins (Fridovich et al., 1998). Cancer has and will eventually evolve both within the body and alongside humanity. Throughout a tumor’s development from initiation to late metastasis, the tumor itself evolves to sustain itself and avoid detection and destruction. Tumors modulate and protect themselves by maintaining complex relationships with immune cells enabling tumor evasion and shielding itself within its own tumor microenvironment (Baghban et al., 2020; De Visser & Joyce, 2023; Kim & Cho, 2022). To tackle such a multifaceted and ever-evolving enemy, any solution needs to be numerous in its methods and modalities as well.

Enabling Multimodality Multimodality in RNA depends on the ability of different genes and elements to be active or translated whilst on the same, singular RNA strand - requiring polycistronic behavior. However, mammalian mRNA molecules are typically monocistronic (Karginov et al., 2017). To enable multimodality, CAR_Ma was designed with the addition of 26S subgenomic promoters (26S), as well as internal ribosome entry sites (IRES), which effectively convert monocistronic RNA to polycistronic RNA (Karginov et al., 2017). hat as many people globally will be able to receive the most advanced therapeutics.

26S is a subgenomic promoter that directs transcription for the subsequent downstream gene, resulting in the formation of mRNA for the single gene (Kamrud et al., 2007). IRES elements are responsible for assembling functional ribosomes at a start codon, allowing for 5’ cap-independent translation initiation, thus allowing for genes downstream of IRES to be translated (Kamrud et al., 2007). Our design adds several 26S and IRES elements in our multimodal RNA, allowing for multiple genes to be present, co-transcribable, and co-translatable.

Transience The transient modality of CAR_Ma focuses on improving therapeutic efficiency while retaining clinical applicability and safety. CAR_Ma itself is a transient system. By relying on transience, we are able to avoid safety risks and risks of malignancies with DNA or viral-based treatments, which may lead to random insertional mutagenesis. Such issues have plagued even the latest CAR immunotherapies, which caused the CAR-T cells to become malignant and are required to come with boxed warnings (U.S. Food and Drug Administration, 2024). Further uses of the transient system facilitate the elimination of immune evasion by tumor cells using CRISPR/Cas systems, or RNAi pipelines. Many cancers hijack systems such as SIRPα-CD47 or PD-1/PD-L1 to evade detection and subsequent targeting by the immune system, preventing clearance of cancerous cells. Knockout of SIRPα expression on macrophages at the genomic level by the CRISPR/Cas systems using Cas9-GFP or Cas12 prevents the SIRPα-CD47 interaction, inhibiting immune evasion via this pathway. By knocking out these evasion pathways, immune cells are able to better recognize their immune targets and destroy the tumors (Ray et al., 2018).

Given knockout by a CRISPR/Cas system occurs at the genomic level, the expression of Cas is not required to be continuously maintained, as a genomic knockout only needs to occur once to result in permanent changes in the expression. Thus, the permanence of genomic changes caused by CRISPR/Cas systems implies the need for a transient modality to avoid unnecessary expenditure of cellular resources. Alternatively, this action can also be carried out via RNAi instead of CRISPR using the release cycle.

Replicative Replicative refers to the need for sustained expression via constant replication. CAR_Ma’s replicative modality contains elements that must be continuously maintained to allow for safe and sustained expression and efficacy of our immunotherapy: a 5th generation nanobody-powered Chimeric Antigen Receptor (CAR), the self-replicating RNA (srRNA) components, and the TMP small-molecule mediated control system.

The self-replicative ability of srRNA components enables persistent and maintained expression of CAR on the cell surface, replenishing receptors lost during receptor-mediated phagocytosis. Continued and tightly controlled expression of the 5th generation nanobody-powered CAR facilitates improved recognition and targeting of hepatocellular carcinoma (HCC) tumor cells, by preventing depletion and stress during receptor-mediated phagocytosis. Simultaneously, the small-molecule mediated control system was implemented to ensure the self-replicating system could be regulated when necessary to avoid depletion of cellular resources. In combination, these elements of CAR_Ma act together to maintain and control the expression of CAR on macrophages.

Release Elements in the release modality consist of RNA molecules that need to be released to confer a function for CAR_Ma, typically when used in conjunction with proteins encoded in other modalities. This includes the guide RNA (gRNA) of CRISPR/Cas systems, as well as short-hairpin RNA (shRNA) acting as an alternative method for SIRPα silencing, based on the principle of RNA interference (RNAi). The release of gRNA enables the targeting of a Cas protein for permanent genomic knockout, while the release of shRNA enables entirely transient transcriptomic silencing of immune evasion (Karim et al., 2018) during the active period of the multimodal RNA. Rather than relying on a permanent method targeting the genome like CRISPR, the use of RNAi and shRNA will allow the system to be completely transient. This means that after its controlled shutdown, the cell will be able to return to a completely native state, enhancing long-term safety and stability for clinical uses. The strategic release of RNA molecules ensures CAR_Ma can effectively modulate gene expression, enabling both transient and permanent solutions to the problems of immune evasion.

Chimeric Antigen Receptor (CAR)

CAR-M vs CAR-T For the next generation CAR-M (CAR_MultiModal Advanced or CAR_Ma) developed by our HKU iGEM 2024 team, the chimeric antigen receptor is expressed on macrophages instead of the most common T-cells, effectively addressing significant challenges in cancer therapy.

One major advantage of this approach is that macrophages can penetrate the tumor microenvironment (TME) more efficiently, overcoming the physical barriers that generate chemokines suppressing T-cell functionality (Bule et al., 2021; Nagarsheth et al., 2017). Additionally, this strategy mitigates the adverse effects of M2-like tumor-associated macrophages (TAMs) and the chemokines that often promote tumor growth and lead to CAR-T immunosuppression (Li et al., 2019; Rodriguez-Garcia et al., 2021). Macrophages can adopt various functional states, enabling them to adapt to the TME and exert anti-tumor effects while reducing immune evasion (Mantovani et al., 2022). This flexibility enhances the persistence of CAR-M cells and improves coordination with other immune cells, resulting in a more robust immune response.

By harnessing the innate capabilities of macrophages for tumor migration and phagocytosis in synergy with the power of vector multi-modality, CAR_Ma not only enhances the potential for a robust anti-tumor response by addressing the limitations of traditional CAR-T systems, but also offers a more adaptable and resilient therapeutic strategy in the fight against cancer.

Self Replicating RNA (srRNA)

TMP Control System

SIRPα Knockout

Transient Gene Modulation

Nanoparticle Delivery

Nanostructured Lipid Carrier In the rapidly evolving field of RNA therapeutics, srRNA has emerged as a powerful tool for the development of various therapeutic cancer vaccines. Clinical trials have demonstrated that srRNA is able to encode tumor-associated antigens such as human epidermal growth factor receptor 2 (HER2), and carcinoembryonic antigen (CEA) (Davis et al, 2021).

In terms of non-viral carriers, lipid nanoparticles were selected as the most effective delivery method due to their superior biocompatibility, reduced cytotoxicity, and enhanced cellular uptake and internalization. Lipid nanoparticles are the most effective due to their ability to prevent endosomal escape– the unique composition of LNPs, specifically the incorporation of pH-sensitive lipids, allows a disruption of endosomal membranes, leading to successful cytoplasmic delivery of our srRNA (Zhao, 2014).

While srRNA has been delivered through a variety of different methods, such as viral vectors, the innovative use of lipid nanoparticles (LNPs) remains a large piece of uncharted territory. Following COVID-19, the remarkable success of mRNA vaccines such as Pfizer and Moderna, exemplifies the excellent biocompatibility of lipid nanoparticle delivery.

However, packaging srRNA is inherently more complex than its mRNA counterparts, due to its unique structural characteristics. To ensure that the nanoparticle we are using is the most biocompatible, while maintaining environmental biodegradability and human safety, we examined a myriad of different lipid-based carriers. Amongst them, we decided that the Nanostructured Lipid Carrier (NLC) fits our project the best. The imperfect crystal model of the NLC, and its amorphous quality allows a higher drug loading capacity, controlled release, and its own edge of higher stability during storage. NLC can be stored at room temperature for at least 8 months and in 4°C for up to 21 months (Gerhardt et al., 2022). Allowing us to simplify logistics and storage making this one of our ideal candidates for CAR_Ma enabling our simplified administration via intravenous injection simplifying and improving accessibility of immunotherapies.

At the current moment, NLCs are currently being used in a multitude of medical trials, such as UV protection cosmetics, dermal drug delivery, and topical nanomedicines (Ferreira et al., 2021). NLC and rvRNAs complexes are successfully designed for Zika vaccines (Erasmus et al., 2018). Similar ingredients were used, of which Tween 80 is replaced with DMG-PEG. They perform similar functions in our case, for the sake of aggregation prevention.

Though NLCs are far more capable of delivery, storage and logistics enabling CAR_Ma simple administration and accessibility. NLCs like other nanoparticle delivery systems are relatively indiscriminate in their transformation targets. Though recipe optimization can better target proportions of the NLCs administered to certain tissue groups, it will be inevitable that other tissues will be transformed. In our case, that could risk complications with incorrect tissues expressing the CAR_Ma system. To optimize this further we functionalized our nanoparticles with anti-CD14 antibodies (Marques et al., 2023) or anti-CD14 nanobodies, to ensure with maximal efficiency that we are able to specifically transfect monocytes and macrophages. Allowing us to maximize efficiency while minimizing unwanted off-target effects, and completing the CAR_Ma package.

Zein Nanoparticle In addition to our work with Nanostructured Lipid Carriers (NLC), our team has innovatively developed zein nanoparticles for the targeted delivery of srRNA into macrophages. Zein, being edible, biocompatible, and biodegradable, stands out as an ideal material for delivering drug and genetic material (Zhang, 2021).

The production process for zein nanoparticles involves transitioning zein from an organic phase (alcohol) to aqueous phase or via altering the pH. This allows aggregation, and rearranges the peptide chains in zein (Zhang, 2021; Dong, 2021). Zein has shown promise in various applications, such as the sustained release of plasmid DNA (pDNA) (Regier et al, 2012). In addition, zein nanofibers have been developed to explore their potential to deliver siRNA for gene silencing in skin fibroblasts (Karthikeyan et al, 2015).

Despite these advancements, aggregation of zein nanoparticles poses a significant challenge, which will limit the viability and shelf-life of zein nanoparticles. To tackle this issue, we added Tween 80, a surfactant that promotes electrostatic repulsion amongst zein nanoparticles. This addition effectively prevents aggregation, and enhances the stability for our formulations.

By leveraging the unique properties of zein, and addressing any potential challenges, we are paving the way for effective, biodegradable, and biocompatible delivery systems for srRNA delivery for CAR_Ma and beyond.

CAR_Ma’s Engineering Cycles

In many ways, the development of CAR_Ma has been very iterative, cycling through several rounds of design, building, testing and learning. Since CAR_Ma is a ground-up rearchitecting of CAR_Ma from HKU iGEM 2023, there are major engineering cycles and smaller iterative ones that have occurred.

Rearchitecting 2023’s CAR_Ma Proof-of-Concept While the work conducted by the HKU iGEM team 2023 was rudimentary, tt allowed us to gain incredible insight into the meticulous design and short-commings of our previous work.

CD3ζ-based CAR From the beginning an iterative immunotherapy was the original goal, in doing so the designs that we used borrowed heavily from one of our advisor’s work with CAR-T, a receptor with a CD3ζ costimulatory domain typically seen with CAR-T therapies. The benefit at the time is that we were quickly able to work with this due to the part already existing with us, unfortunately it was found that with macrophages CD3ζ was a costimulatory domain that did not greatly improve phagocytosis. Our testing, review of literature by Morrissey et al. (2018) and feedback from our PI and others from 2023 iGEM Giant Jamboree. Learning from this undersight, we completely rearchitected our CAR based on the work by Morrissey et al. (2018), integrating CD19-FcγR Tandem and MegF10 costimulatory domains into our CAR enhancing phagocytosis, trogocytosis and antigen presentation resulting in a far more effective system.

Overwhelming cellular resources, IFN-γ overexpression and TMP shutdown Besides the core of our CAR being based on costimulatory domains we integrated IFN-γ as an armouring factor to reinforce our CAR_Ma cells and repolarize tumor associated macrophages (TAMs) disrupting the stability of the tumor microenvironment. Another system we integrated was a small-molecule based shutdown system based on trimethoprim (TMP) to terminate the expression upon the completion of the treatment regime. In our initial experiments from 2023, there were difficulties with cloning our control system into our plasmids, delaying their completion. We also noted that CAR_Ma cells demonstrated an impressive effect in initial tests but exhibited shorter than expected lifespans. After further investigation, and discussion with our PIs and their colleagues we were able to determine that the CAR expression with srRNA if left uncontrolled would quickly runaway and induce excessive stress on cellular machinery and resources. One PI also noted that IFN-γ in small doses resulted in the armoring of CAR-M and repolarization TAMs, however the sustained expression via our self-replicating RNA (srRNA) vector would lead to severe downstream immune responses which could risk patient health.

As it turns out, the solution to these problems all could be solved by our small-molecule shutdown system. After these learnings, the shutdown system was broken-down and rearchitected completely into a system that allows for variable control. In a recent paper on the in vivo validation of the TMP-based shutdown system on mice, Cafferty et al. (2021) found that there was a distinct curve relating the dose of TMP given to the strength of expression. After amending our cloning scheme to streamline the integration of the shutdown system’s functional units we designed our shutdown system to regulate both the expression of CAR but also the replication of the srRNA itself. Our shutdown system initially designed for termination in case of emergencies and completion of the treatment regime was now evolved to allow for the control of the CAR expression and IFN-γ release, by simply pairing each dose of CAR_Ma with a corresponding dosage of TMP. With this, our new CAR_Ma cells are able to retain overall effectiveness, but gain increased longevity and reduce immunological side-effects with tighter control.

In vivo vs Ex vivo: Delivery, CRISPR and Gene Modulation When 2023’s proof-of-concept moved away from Sendai viral delivery to self-replicating RNA based vectors, the auxiliary engineering aside from CAR expression was not fully adapted to the new system. The importance of mitigating immune evasion via pathways such as SIRPα/CD47 have been well established in enhancing phagocytosis of tumor cells (Ray et al., 2018), but since common methods currently of delivering CRISPR operations for therapeutics were delivered ex vivo such as those via ribonucleoprotein (RNP) complexes, CAR_Ma cells were actually engineered with a two-step process that required the knockout of SIRPα first by the electroporation of an RNP, then the transformation with srRNA for CAR expression also via electroporation. Though effective for use in the laboratory, this negated a significant advantage of using RNA as a vector as the cell must still be engineered ex vivo. This was acceptable for a proof-of-concept, but to not only be a viable but superior therapeutic, this would be insufficient.

Ultimately, the Holy Grail of CAR_Ma would be in vivo expression of CAR but also the in vivo gene editing of SIRPα via CRISPR. When we rearchitected CAR_Ma, we went even a step further by integrating both these functions on to the same RNA molecule dramatically simplifying production by only requiring one template but also vastly simplifying packaging and screening by allowing for a homogenous RNA payload. The advancement we required was born out of engineering our self-replicating RNA and using the functional blocks to allow for transient expression of Cas proteins, sustained expression of CAR and the release of small non-coding RNA such as gRNA on a unified multi-modal RNA vector. Once this breakthrough was achieved, we needed a vector to preserve the goal of in vivo delivery. As the world recovered from the COVID-19 pandemic, inspiration struck. The mRNA vaccines used for COVID-19 used lipid nanoparticles for in vivo delivery. When discussing with our PIs and collaborators in microfluidics, we engineered two cutting edge nanoparticles to package our system: A nanostructured lipid carrier and a zein-based protein nanoparticle. Allowing us to achieve the goal of an in vivo immunotherapy, while setting the stage for a large array of gene therapies and immunotherapies by combining CRISPR, our multi-modal RNA and nanoparticle delivery systems.

Minor Iterations enhancing CAR_Ma Aside from breakthroughs from the rearchitecting of 2023’s CAR_Ma, there were several crucial iterations done through minor engineering cycles during the experimentation done this year.

CRISPR vs RNAi When originally conceived, CAR_Ma and the 2023 proof-of-concept were built to use CRISPR. Since knockout studies that facilitated the knowledge of tumor evasion, knockout improving phagocytosis were all using CRISPR and even modern gene therapeutics based upon CRISPR it seemed as the obvious choice. Especially since the tool sets to use CRISPR were more comprehensive and robust. However, a tenet of CAR_Ma was transience. With the system itself having the ability to be modulated, shutoff and even removed. The persistence of the CRISPR knockout long-term broke this tenet and further posed significant risks downstream. Mainly, since CRISPR knockouts are permanent the activity of the knocked out gene in our case would remain off even after the CAR_Ma system expires or is shut down via TMP, this could lead to severe risks and complications where these cells no longer ignore friendly cells leading to an autoimmune risk. Further, by acting at a genomic level CRISPR knockout would only have a complete shutdown of SIRPα, lacking any control or variability.

Countering this, we moved away from genomic gene modulation towards transcriptomic regulation instead. By leveraging RNA interference, the SIRPα gene would remain untouched producing SIRPα RNA transcripts. By transcribing shRNA from our multi-modal RNA vector, we are able to trigger RNA interference causing the specific degradation of targeted SIRPα RNA transcripts, effectively silencing the gene expression transiently. Furthermore, by leveraging our TMP-modulated variable switch, we are able to adjust the expression of the shRNA effectively controlling the strength of the SIRPα silencing granting the system even more flexibility.

Unfortunately, the shRNA sequences are unable to directly be added onto our RNA release system. When initially designed, our RNA release system had a severe deficiency in that the RNA-dependent RNA polymerase from the Venezuelan equine encephalitis (VEE) virus replicase that powers the CAR_Ma inherently has capping and tailing capabilities. With our Cas9-mediated knockout, the gRNA was expressed enough to allow for the knockout to occur. But when moving to our Cas12a multiplexed gRNA and especially with shRNA, we noticed that the knockouts were unsuccessful. Upon further investigation we discovered the capping and tailing capability of the VEE polymerase. We quickly adapted, designing self-cleaving ribozymes to the 5’ and 3’ ends of the shRNA. By adding a hammerhead ribozyme to the 5’ and a HDV ribozyme to the 3’ end, we are able to perfectly preserve the sequence of the gRNA or shRNA during the subgenomic release of the non-coding RNA, furthermore the ribozyme sequences will not cleave as effectively on the parent multi-modal RNA due to the length and structural characteristics of the longer RNA molecule inhibiting effective cleavage. The resulting system allows gRNA or shRNA to be replicated, and its subgenomic release sustained while preserving the exact sequence needed for maximal efficiency for CRISPR or RNAi operations.

PCR, Cloning and Transformation Through designing the complexities and intricacies of CAR_Ma, there were a lot of areas for errors to occur. The complexities required a cloning system with high-specificity and the ability to assemble plasmids from over 8 fragments. Eventually we landed on cloning with NEBuilder as it was a system that was both accessible to us and matched our needs perfectly. However using NEBuilder would require designing over 30 different gene fragments to be assembled in various ways along with over 100 different primers.

Cloning and assembly of fragments was laborious and required frequent use of PCR, oftentimes requiring optimization as our PCRs would be run in gels, sometimes resulting in mispriming, smearing, or in the worst case lacking amplification. By optimizing PCR recipes and run operations: Adjusting amount of template, primers, including additives such as DMSO. Running touchdown, using hot-start polymerases, running longer extensions. We were able to optimize a majority of our PCR processes to create an optimal amount of fragment for cloning. NEBuilder itself also often required optimization, adjusting incubation times or fragments to be in equivalent molarities.

The final step in our cloning would be transformation into cloning strains of E. coli. We found that this process has been the most troublesome throughout all of our experiments. Initially we worked with E. coli DH10b for its capability to work with larger plasmids, however we found that they would never be any transformants. After optimizing upstream processes and verifying that our plasmids would successfully assemble, we narrowed down the process to the transformation. Eventually, we were able to diagnose that the large plasmid would be too heavy on cellular resources, thus requiring an extended recovery time of 2 hours to successfully express the relevant antibiotic resistance gene for selection, thus we were able to finally get transformants. However, after miniprepping our transformants and sending our plasmids for sequencing, we noticed significant portions of our plasmid were missing. Including CAR or shRNA, essential parts of CAR_Ma. We were able to soon diagnose that the repeated sequences, especially with shRNA were toxic to the E. coli resulting in the cell excising the DNA. To remedy this, we moved away from E. coli DH10b and used E. coli Stable, which provided us with the ability to clone toxic and repeated sequences. Eventually, we were able to optimize our overall cloning and transformation process to enable us to get the required plasmids we needed to develop and characterize CAR_Ma.

CAR_Ma Parts List

Basic Parts Kozak sequence - BBa_K4344028
Signal peptide BBa_K2549044
CD8 hinge: BBa_K5017003
CD28 transmembrane domain BBa_K4803006
CD19 BBa_K5062003
FcyR BBa_K5062004
MegF10 Costim - BBa_K5062005
Anti-gpc3 nanbody BBa_K5062000
Anti-GPC3 scFv BBa_K5062001
DDd BBa_K4696007
L7Ae - BBa_K3113009
2x kTurn - BBa_K4696005
P2A - BBa_K1442039
Ifn-gamma BBa_K4696020
mCherry XL BBa_K4241792
5' Hammerhead Ribozyme BBa_K5062009
3' Hepatitis D Virus Ribozyme BBa_K5062010
SIRPa shRNA BBa_K5062011
PD1 shRNA BBa_K5062012
SIRPa flank sequence BBa_K5062013
PD1 flank sequence BBa_K5062014
26s BBa_K4696004
-26s BBa_K5062033
nsP10 BBa_K5062031
nsP14 BBa_K5062029
Cas9 gRNA BBa_K5062020
Cas9 gRNA flank sequence BBa_K5062032
IRES BBa_K5062038
Enhanced IRES BBa_K5062037
nsP1 - BBa_K4696000
nsP2 - BBa_K4696001
nsP3 - BBa_K4696002
nsP4 - BBa_K4696003
Cas12a gRNA flank sequence - BBa_K5062034
DR1 - BBa_K5062023
DR2 - BBa_K5062025
DR3 - BBa_K5062027
SIRPa gRNA - BBa_K5062022
PD-1 gRNA - Part:BBa_K5062024
VTCN1 gRNA - Part:BBa_K5062026
STAB1 gRNA - Part:BBa_K5062028
AsCpf1 (Cas12a) - BBa_K5062021
Puromycin resistance - BBa_K4696009

Composite Parts DD-L7Ae - BBa_K5062002
CD19/ FcyR Tandem - BBa_K5062006
5th Generation NanoCAR - BBa_K5062007
5th Generation CAR - BBa_K5062008
SIRPa Ribo-shRNA - BBa_K5062016
PD-1 Ribo-shRNA - BBa_K5062017
SIRPa/PD1 Multiplex shRNA - BBa_K5062018
Cas9-GFP - BBa_K5062030
Cas9 gRNA Ribo - BBa_K5062039
Multiplex Cas12a gRNA - BBa_K5062045
nsP123 - BBa_K5062046

Multi-modal RNA CAR_Ma - BBa_K5062047
CAR_Ma shRNA Multiplex - BBa_K5062048
CAR_Ma Cas9 - BBa_K5062049
CAR_Ma Cas12a Multiplex - BBa_K5062050

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