Current approaches to the targeted delivery of therapeutics such as gene-silencing nucleic acids have to date tended to focus on a single-target cell receptor, but truly personalized treatments for many diseases, such as cancer, will require the ability to target potentially multiple cell types and their receptors, which will vary between individuals. This has been technologically challenging, not least from a design and production perspective.

Researchers at Tel Aviv University (TAU) have now developed a flexible, self-assembling small interfering RNA (siRNA) carrier platform that they claim can easily be customized to target any cell receptor. The technology, which doesn't rely on chemical conjugation, is based on a membrane-anchored lipoprotein, or linker (anchored secondary scFv enabling targeting, or ASSET), which is incorporated into siRNA-loaded lipid-based nanoparticles (LNPs), and which binds to the Fc region of antibodies.

“The siRNA delivery targeted carriers constructed today hone in on specific cells and require chemical conjugation of the targeting agent,” says lead researcher Dan Peer, Ph.D., of the Laboratory of Precision Nanomedicine at TAU's School of Molecular Cell Biology and Biotechnology. “The new platform is based on biological affinity, a self-assembling approach that can be translated to target an endless number of diseases and conditions.”

In a report in Nature Nanotechnology, the mutidisciplinary TAU team, together with colleagues at Harvard Medical School and Integrated DNA Technologies, describe use of the platform to deliver cell-targeting siRNAs that improved disease symptoms in a mouse models of inflammatory bowel disease (IBD), and improved survival in a xenograft model of mantle cell lymphoma. In their paper, entitled, “A Modular Platform for Targeted RNAi Therapeutics,” the researchers claim that the platform “represents a milestone in the development of precision medicine.”

Major progress has been made in developing targeted siRNA delivery carriers that use monoclonal antibodies to target cell-surface receptors, but translating such technologies into the clinic hasn’t been easy, the researchers write. This is partly because of “the massive development and production requirements and the high batch-to-batch variability of current technologies, which rely on chemical conjugation.”

“The challenge has been how to direct certain therapies designed to manipulate genes of interest in specific cells without developing a specific drug carrier for each specific cell type,” adds Nuphar Veiga, who is co-first author of the paper and a researcher in professor Peer's laboratory. “It would be very costly and impractical to develop millions of different drugs to treat every specific cell type and specific gene. Rather, the focus should be on developing a nucleic acid-based tool to manipulate gene expression through a simple, constant exchange.”

Unlike current technologies, the new self-assembly modular platform has been designed to allow the creation of what the researchers suggest is a “theoretically unlimited repertoire” of siRNA-targeted carriers. “Because its construction relies on affinity interactions, there's no need to introduce chemical conjugation optimizations for the method to function,” Prof. Peer notes. “Linkers are stuck in the nanoparticle membrane and bind to a fixed region of any antibody of the same isotype. This affords safe passage to a theoretically unlimited selection of carriers targeting distinct cell-surface receptors.”

To generate the siRNA carriers, the LNPs are noncovalently coated with targeting antibodies via the recombinant, membrane-anchored ASSET lipoprotein, which interacts with the antibody Fc domain. In contrast with chemically conjugated antibodies, “which are randomly oriented so only a fraction is functional,” the TAU team’s targeted siRNA-loaded LNPs, or TsiLNPs, ensure optimal antibody function, because the antibody is bound through its Fc domain, which leaves the variable domain exposed for ligand binding. “Our targeting strategy involves a simple antibody substitution without any recalibration or optimization, which preserves the high affinity of the antibody in the nanoparticle and eliminates its phagocytosis by scavenger cells.”

The researchers first confirmed that TsiLNPs can bind to and are taken up by cells bearing the antibody-targeted receptor. They then demonstrated that TsiLNPs could knock down their target genes in hard-to-transfect lymphocytes, in vivo. Switching which type of rat antibody (Rig) was bound to the construct meant it was possible to change which cells were targeted. “Thus, simply switching the RIg used for targeting directed LNPs in vivo into cell-surface receptor-defined subsets of lymphocytes with precision,” they state.

In a final set of in vivo therapeutic experiments the researchers tested the ability of of TsiLNPs to knock down tumor necrosis factor-α (TNFα) production by macrophages in a mouse model of IBD. The results suggested that siRNA delivery “significantly and dramatically” reduced major symptoms of disease. “One can easily obtain fast results using these targeted carriers,” says Prof. Peer. “The mice exhibited far less inflammation, which suggests the possibility of promising new IBD therapeutic opportunities.”

Then, to evaluate potential use of the system for cancer therapy, the researchers tested the TsiLNP platform in a mouse mantle cell lymphoma (MCL) xenograft model, demonstrating that treatment led to cancer cell death and signficantly improved survival. 

“Here we showed the versatility of this platform for antibody-directed delivery and knockdown in hard-to-transfect immune cells using eight different targeting antibodies (against CD44, CD34, Ly6C, CD3, CD4, CD25, CD29 and Itgb7) and showed its therapeutic effectiveness in a DSS [dioctyl sodium sulfosuccinate] colitis model and in MCL xenografts,” the authors write.

“Adapting this flexible platform for human use will enable personal therapy for cancer patients in which the targeted antibody can be adjusted according to each patient’s cancer cell-surface marker expression. The platform could also allow the use of more than one targeting antibody to reduce the chance of cancer resistance and relapse due to receptor down-modulation.”

In effect, the siRA delivery platform “can be adjusted for each patient to target a potentially endless number of receptors,” Prof. Peer states. “It's flexible enough to be easily customized to target any cell subset and to knock down any gene of choice. We think it has tremendous research and therapeutic potential.”




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