Periodic Reporting for period 1 - nanoVAST (nanoVAST: a novel, non- viral LNP for precision payload delivery of genome editors and other cargo)
Berichtszeitraum: 2022-10-01 bis 2024-03-31
Numerous diseases are attributed to faulty genes - from genetic disorders such as in muscular dystrophy to accumulated mutations such as in cancer. Gene therapies span a wide range of indications to correct these faulty genes. These include the introduction of a functional gene to replace a faulty gene or combat a specific disease. More recently, genome and RNA editing introduced the possibility of correcting the patient's own genes or regulating their expression. However, the potential of these gene therapies is not yet fully realized due to limitations in current gene delivery technologies.
Viral vectors and lipid nanoparticles (LNP), such as those in the vaccines used to combat the SARS-CoV-2 pandemic, are currently used and are being developed for the delivery of gene therapies. Though proven to be highly effective, each of these have their own limitations. Viral vectors are limited by immunogenicity where patients may have or develop antibodies against the vector limiting their broad use and effectiveness. There is also the perceived danger of permanent integration of the gene therapy into the patient’s genome. Viral vectors can only carry limited gene lengths precluding the delivery of larger sized genes. LNPs are less immunogenic and have a higher gene length capacity compared to viral vectors. However, they are not readily targetable which is critical for gene therapies that require precise delivery to specific organs. Furthermore, LNP gene therapy delivery is inefficient due to the cargo often being trapped within endosomal compartments of the cell where they are unable to function. Our project developed a targeted gene therapy delivery vehicle that is neither viral or LNP-based and would provide advantages over these.
We worked on trypanosome-derived synthetic extracellular vesicles (EV), which we call nanoVAST. These nanoVAST vesicles have a dense protein coat consisting of a single variant surface glycoprotein (VSG), and we have shown that this unique coat provides unique opportunities for gene therapy delivery. We developed methods to produce, load, and attach targeting molecules to these vesicles. We focused on developing a modular targetable delivery vehicle as targeting as this would be a huge leap forward for gene therapy delivery. We designed nanoVAST to home in and deliver their therapeutic cargo to the intended organ. Doing so should lower dose, improve efficacy, and reduce side effects of gene therapies. Moreover, a precision delivery tool would unlock the therapeutic potential for more diseases than are currently considered druggable via gene therapy, including but not limited to many cancers and genetic disorders.
Viral vectors and lipid nanoparticles (LNP), such as those in the vaccines used to combat the SARS-CoV-2 pandemic, are currently used and are being developed for the delivery of gene therapies. Though proven to be highly effective, each of these have their own limitations. Viral vectors are limited by immunogenicity where patients may have or develop antibodies against the vector limiting their broad use and effectiveness. There is also the perceived danger of permanent integration of the gene therapy into the patient’s genome. Viral vectors can only carry limited gene lengths precluding the delivery of larger sized genes. LNPs are less immunogenic and have a higher gene length capacity compared to viral vectors. However, they are not readily targetable which is critical for gene therapies that require precise delivery to specific organs. Furthermore, LNP gene therapy delivery is inefficient due to the cargo often being trapped within endosomal compartments of the cell where they are unable to function. Our project developed a targeted gene therapy delivery vehicle that is neither viral or LNP-based and would provide advantages over these.
We worked on trypanosome-derived synthetic extracellular vesicles (EV), which we call nanoVAST. These nanoVAST vesicles have a dense protein coat consisting of a single variant surface glycoprotein (VSG), and we have shown that this unique coat provides unique opportunities for gene therapy delivery. We developed methods to produce, load, and attach targeting molecules to these vesicles. We focused on developing a modular targetable delivery vehicle as targeting as this would be a huge leap forward for gene therapy delivery. We designed nanoVAST to home in and deliver their therapeutic cargo to the intended organ. Doing so should lower dose, improve efficacy, and reduce side effects of gene therapies. Moreover, a precision delivery tool would unlock the therapeutic potential for more diseases than are currently considered druggable via gene therapy, including but not limited to many cancers and genetic disorders.
We worked on optimizing the synthetic production of nanoVAST from Trypanosoma brucei. We needed a scalable method of producing nanoVAST as natural production of trypanosome EVs is low. We further developed our sonication-based production method that shears the cleared trypanosome membranes and spontaneously produces 100-150 nanometer (nm) sized vesicles with an intact protein coat. Our method can now yield high quantities of well-purified nanoVAST particles, a process that we foresee could be scalable for potential clinical trials.
Further, we were able to load an RNA cargo simultaneously during nanoVAST production. We loaded a guide RNA for RNA editing and a green fluorescent protein (GFP) mRNA for gene expression. We used these RNA tools to measure the efficiency of cargo loading and delivery by quantitative PCR (qPCR). Further improvements to the loading efficiency are currently in development, although even with relatively “low” amounts of loaded cargo, we were able to detect RNA delivery to target cells in vitro. Furthermore, we confirmed the hypothesis that nanoVAST can deliver cargo through direct fusion with the target cell membrane. This avoids endosomal entrapment, a weakness for LNP based delivery technologies, by releasing the RNA cargo directly into the cell cytosol. We observed this directly through images of the fusion event with electron microscopy, as well as by interrogating the different dynamics of RNA reporter delivery in cells compared to a lipid-based delivery vehicle.
For directed cell targeting, we used a B-cell targeting antibody fragment (similar to a conventional “nanobody”). Using the sortase enzyme, we were able to attach the anti-CD19 nanobody onto the protein coat of nanoVAST at high efficiency. We used an in vitro model with Ramos B cells to illustrate that the coupling of this nanobody to the nanoVAST substantially improved B-cell targeting. nanoVAST had a higher preference for the Ramos B cells compared to the untargeted nanoVAST. This together with the ease of attaching the targeting molecule highlight the potential targetability of nanoVAST.
Further, we were able to load an RNA cargo simultaneously during nanoVAST production. We loaded a guide RNA for RNA editing and a green fluorescent protein (GFP) mRNA for gene expression. We used these RNA tools to measure the efficiency of cargo loading and delivery by quantitative PCR (qPCR). Further improvements to the loading efficiency are currently in development, although even with relatively “low” amounts of loaded cargo, we were able to detect RNA delivery to target cells in vitro. Furthermore, we confirmed the hypothesis that nanoVAST can deliver cargo through direct fusion with the target cell membrane. This avoids endosomal entrapment, a weakness for LNP based delivery technologies, by releasing the RNA cargo directly into the cell cytosol. We observed this directly through images of the fusion event with electron microscopy, as well as by interrogating the different dynamics of RNA reporter delivery in cells compared to a lipid-based delivery vehicle.
For directed cell targeting, we used a B-cell targeting antibody fragment (similar to a conventional “nanobody”). Using the sortase enzyme, we were able to attach the anti-CD19 nanobody onto the protein coat of nanoVAST at high efficiency. We used an in vitro model with Ramos B cells to illustrate that the coupling of this nanobody to the nanoVAST substantially improved B-cell targeting. nanoVAST had a higher preference for the Ramos B cells compared to the untargeted nanoVAST. This together with the ease of attaching the targeting molecule highlight the potential targetability of nanoVAST.
The results of our work demonstrate that nanoVAST could be used for gene therapy delivery. The unique trypanosome origin of nanoVAST gives it two differentiating characteristics that could be exploited for targeted gene therapy. (1) nanoVAST particles are covered in a dense variant surface glycoprotein (VSG) coat derived from the trypanosome host that provides a repeating array where single or multiple different targeting molecules can be attached across its surface. (2) We have demonstrated direct fusion of the nanoVAST particle to its target cell to directly release the genetic payload into the cytosol, avoiding entrapment of the payload in endosomal compartments. These two traits in combination make this delivery system a strong candidate for targeted gene therapy delivery. However, this is still only potential. Further work needs to be done regarding the immunogenicity or toxicity of these particles. We have not observed any obvious indications of toxicity in vitro although preclinical immunogenicity and toxicity screens in animal models will be required. We have also yet to demonstrate the effectiveness of delivery in in vivo animal models, which will also be pursued in the future. Importantly, we have laid the groundwork for translation and commercialization of this research through the filing of a patent (WO2023175016A1) on the production and use of nanoVAST for therapeutic delivery.