Targeted Delivery of Self-Amplifying RNA Genes

As a result of billions of years of evolution, what viruses do better than anything else -- better than anything else they do, or any thing else does -- is to protect and deliver genes to specific (targeted) cells. This is why it is impossible not to consider their use as gene delivery systems for medical purposes. To avoid the complications and potential dangers of using "attenuated" or "inactivated" viruses, however, we pursue a research program that involves the design and synthesis -- from purified components in test tubes -- of virus-like particles that contain genes of interest in (single-stranded) RNA form. The RNA form is essential for enabling spontaneous self-assembly of the gene into a virus-like particle when mixed with the right viral capsid protein; this wouldn't be possible with (double-stranded) DNA. For self-assembly purposes, the "right" capsid protein is one that will efficiently package any RNA, independent of its sequence, as long as it isn't too long or too short. We have found that capsid protein from cowpea chlorotic mottle virus (CCMV) or brome mosaic virus (BMV) does this job. The other advantage of RNA genes is that it is possible to work with them in messenger-sense and self-amplifying form, enabling them to be strongly replicated in the cytoplasm of targeted host cells before being translated into proteins of interest.

Design and synthesis of self-replicating forms of mRNA (replicons)

We design and synthesize self-replicating forms of messenger RNA coding for reporter and therapeutic genes, to be packaged into CCMV or BMV virus-like particles by in vitro reconstitution. The basic idea is to use the RNA-dependent RNA polymerase (RdRp) of a positive-sense/ready-to-translate viral genome. The 1^st molecule (RNA1, ~3000nt) of the RNA genome of Nodamura virus, for example, is directly translated to give an RdRp that strongly replicates it. By adding a gene of interest to RNA1, that gene – coding for a reporter like EYFP or luciferase, or a therapeutic protein like a viral or cancer antigen – is strongly replicated along with the RdRp gene and translated to give the target protein of interest. The resulting construct – a single-molecule replicon – is still short enough (~4000nt) to be in vitro reconstituted in CCMV or BMV VLPs, which can then be functionalized by targeting ligand or wrapped in lipid bilayer and used for gene delivery.

COVID-19 Vaccine in the Form of Self-Amplifying mRNA Reconstituted in Virus-Like Particles Conjugated with Targeting Ligands

The highly effective Moderna and Pfizer vaccines are essentially “just” mRNA molecules encoding the SARS-2 spike protein, complexed with cationic lipid and surfactant in a “lipid nanoparticle” (LNP). We are working on a vaccine particle that differs in several significant ways: (1) instead of mRNA we use self-amplifying (sa) – replicon – mRNA, i.e., sequence encoding the spike protein and that is fused with the RNA replicase gene of Nodamura virus; (2) instead of the relatively unstable LNP formulation, we in vitro package our replicon into a perfectly monodisperse, RNase-resistant, thermally-stable, BMV VLPs; and (3) we functionalize this VLP with protein ligand that targets a receptor on cross-presenting dendritic cells, to maximize antigen presentation by those cells. In collaboration with Dr. Otto Yang in the Department of Infectious Diseases in the UCLA School of Medicine, we are beginning to test these vaccine VLPs against Moderna LNPs. Specifically, we use ELISPOT assays to quantify the proliferation of (gamma-interferon-secreting) spike-protein-specific killer T cells from COVID-19 convalescent patients, following their incubation with antigen-presenting cells (from the same patient) that have been activated by the vaccine particles.

3D ("Organoid") versus 2D (Cell Culture) Growth of Viruses and Replicons

There are big differences between how a mammalian virus undergoes its life cycle in vitro and in vivo. By “in vitro” here we mean in sub-confluent-monolayer cell-culture plates, and by “in vivo” we mean in the context of its natural host animal. In the latter case the gene regulation network of the infected cell is strongly influenced by a wide range of physical and signaling interactions between cells, and of course by the systemic immune system of the host. A huge advantage of the former case is that one can control many important “input” parameters such as the “multiplicity of infection”, and quantify directly many “output” characteristics such as “burst size”, etc. A middle ground is provided by 3D reconstructions (“organoids”) of tissue which incorporate much of the three-dimensional cell-cell organization and yet still allow for control of viral input and output and for direct imaging and quantification. We are collaborating with Dr. John Mellnik at Path BioAnalytics – a biotech company specializing in lung organoids – in an effort to compare the activity of persistent Sindbis replicons in 2D and 3D cell culture.

In vivo T-cell Therapy: Delivery of VLP-Protected CAR and TCR mRNA to Killer T Cells

Chimeric antigen receptor (CAR) and T-cell receptor (TCR) therapy are among the most exciting and promising approaches to cancer immunotherapy. The idea is to provide a patient directly with the particular antigen-specific cytotoxic T cells needed to kill their cancer. This is done by ex vivo transformation of the patient’s T cells, i.e., by extracting T cells from the body, and then expanding/proliferating them, transforming them with gene-integrating vectors like lentiviruses to induce expression of the desired CARs or TCRs, and finally infusing them back into the body. With Dr. Otto Yang in the UCLA School of Medicine, we are pursuing a combination of in vitro and in vivo approaches to T-cell therapy which avoid these costly and risky procedures. More explicitly, we: synthesize in vitro the mRNA encoding a particular CAR or TCR; package it in vitro into virus-like particles involving one copy of the RNA molecule inside a 26-nm/icosahedrally-symmetric/180-subunit protein shell; and conjugate these VLPs with an antibody against the CD3 protein subunit in T-cell receptor complexes. These particles will be taken up by T cells in vivo and transform them into ones expressing the mRNA-encoded CAR or TCR. We are presently working on demonstrating this by incubating them with purified T cells, using a CAR that is specific against the CD19 antigen over-expressed in cancerous B cells, with downstream flow-cytometry quantification of CAR expression and cell-killing assays with CD19-transformed cells. These experiments will be followed up with mouse studies in the Yang lab.

Delivery of microRNA replicons as a pancreatic cancer treatment

MicroRNAs have been actively studied for their potential application to treatment of a wide variety of diseases – both in the context of gene regulation at the translational level, and of transcriptional silencing and direct interaction with regulatory proteins. Using RNA replicase genes from positive-sense RNA viruses, we have constructed self-replicating forms of microRNAs in analogy with amplification of mRNAs in our gene and vaccine delivery work. The miRNA sequence of interest is flanked by self-cleaving ribozymes and added to the replicase gene of the Nodamura virus, which is directly translated to generate an RNA-dependent RNA polymerase that replicates it strongly along with the doubly-self-cleaving ribozyme cassette containing the microRNA. Transfection of mammalian cells with replicons of this kind results in the generation of as many as a million microRNAs per cell. In the case of microRNA-34a the biological activity of microRNA amplified in this way is demonstrated by the essentially complete apoptosis of cultured prostate cancer cells transfected with the replicon.

In collaboration with Dr. Stephen Pandol and Mouad Edderkaoui (Los Angeles, Cedars Sinai Hospital)

In vitro reconstituted CCMV VLPs for delivery of self-amplifying cancer antigens

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. We developed a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying (“replicon”) mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers – CD80, CD86 and MHC-II – and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction [1].

In collaboration with Boehringer-Ingelheim Pharmaceuticals (Ridgefield, USA, Biberach, Germany, and Vienna, Austria)

[1] Biddlecome, A., Habte, H. H., McGrath, K. M, Sambanthamoorthy, S., Wurm, M., Sykora, M. M., Knobler, C. M., Lorenz, I. C., Lasaro, M., Elbers, K., Gelbart, W. M. (2019) Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles. PLoS ONE, 14 (6): e0215031.

In vitro reconstituted TMV VLPs for delivery of chemo and Sindbis RNA therapeutics as an ovarian cancer treatment

We are making in vitro self-assembled virus-like particles (VLPs) made from ssRNA and the capsid protein from tobacco mosaic virus (TMV) instead of from CCMV. TMV was the very first virus to be reconstituted from its purified components, and – as with CCMV – its capsid protein is capable of packaging heterologous RNA as long as the TMV RNA “packaging signal” is inserted into the foreign RNA to ensure the nucleation of a capsid. The capsid that forms is a long hollow/helical cylinder of protein instead of an icosahedral spherical shell. Because the curvature (reciprocal radius) of the cylinder remains constant independent of its length, there is no limit to the length of heterologous RNA than can be packaged in this way. This fact allows us – in contrast to the situation with CCMV or BMV VLPs – to accommodate replicons of any length, and to therefore use RdRp genes from mammalian viruses like Sindbis instead of being restricted to RdRps from insect viruses like Nodamura that happen to work – but, not surprisingly, not as well as those from mammalian viruses – in mammalian cells. Further, the packaged RNA is accommodated inside the 7nm-thick protein shell itself, leaving the 4nm-diameter hollow interior available for loading of other therapeutics. Specifically, in collaboration with the Steinmetz group at UCSD we are preparing and packaging RNA replicons derived from the ~7600nt-long RdRp genes of Sindbis, to which we add genes of interest along with the TMV packaging signal; the hollow interior is then loaded with a chemoagent like cis-platin, whose positive charge is opposite to that of the inside surface of the capsid.

In collaboration with Prof. Nicole Steinmetz's group (NanoEngineering, UCSD)

Defective interfering RNA (DI RNA) as a Yellow Fever anti-viral

We are developing an RNA-based therapy for Yellow Fever Virus (YFV) via the construction of defective-interfering (DI) RNA molecules. The DI RNAs in this case involve specifically engineered deletion mutants of the yellow fever genome that are noninfectious yet still replication-competent; they are replicated by the viral RdRP to the detriment of the genome itself, thereby lowering the viral load. While many viruses, e.g., influenza, have naturally-occurring DIs that allow for a lower-grade/more-persistent infection, YFV does not, so we need to identify one by rational design in the lab.

Targeted delivery of gene therapies through protein fusions and chemical conjugation of ligands to reconstituted virus-like particles

We are designing, expressing, and purifying recombinant proteins that are fusions of CCMV or BMV capsid protein (CP) with antibodies and other targeting ligands, for purposes of enhancing the targeting specificity of VLP self-amplifying-gene delivery systems. Aside from the usual challenges of assuring strong bacterial expression in soluble fractions, the fusion inserts must be done in a way that doesn’t interfere with the in vitro reconstitution/self-assembly of ssRNA-VLPs. In parallel, we are working on a set of alternative approaches that involve making wildtype-CP VLPs and then functionalizing them with protein ligands using a variety of conjugation- and click- chemistry techniques. Additionally, we are preparing CPs with sortase insertions for facilitating the presentation of arbitrary targeting proteins on the outside surfaces of VLPs.

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