The use of gene-regulating microRNAs (miRNAs) for treating cancer is a promising concept, but in practice the approach has been hobbled by a lack of efficient delivery vehicles. miRNAs are subject to enzyme attack in the bloodstream, so traditional delivery approaches have involved packaging the short RNA strands in protective particles such as lipid-encapsulated nanoparticles. Unfortunately, such delivery vehicles can be toxic or are too big to efficiently penetrate the tumor microenvironment. 

A team of researchers at Purdue University has now developed an miRNA delivery technique that does away with protective delivery vehicles completely. Instead, the approach couples a “naked” miRNA molecule to folate (vitamin B9) to create a FolamiR conjugate that targets the folate receptor, which is overexpressed on many types of solid and hematologic cancers, but is expressed at insignificant levels on normal cells.

In today’s Science Translational Medicine, Purdue University’s Andrea L. Kasinski, Ph.D., and colleagues report on the development of a FolamiR conjugate based on the clinically tested miRNA-34a (miR-34a). Their initial in vitro experiments showed that the FolamiR-34a conjugate was selectively taken up by triple-negative breast cancer cells that overexpressed the folate receptor. In subsequent in vivo tests, FolamiR-34a shrank tumors in immunocompromised mice bearing breast cancer xenografts and in immunocompetent mice with an aggressive form of non-small-cell lung cancer. There was no evidence of any toxicity at the doses used. The team’s results are published in a paper entitled “FolamiRs: Ligand-Targeted, Vehicle-Free Delivery of MicroRNAs for the Treatment of Cancer.”

“The evidence presented here supports a new method for vehicle-free miRNA delivery,” they write. “This approach has completely removed the toxic delivery vehicle without impairing miRNA delivery or activity. It is, thus, highly plausible that miRNAs could be delivered at high quantities and in the absence of unwanted toxicity to treat various diseases such as cancer.”

The team wasn’t 100% convinced that the technology would generate such positive results, admits Dr. Kasinski, who is the William and Patty Miller Assistant Professor of Biological Sciences at Purdue University’s department of biological sciences. “We were not entirely optimistic that it would work, and as you can imagine were excited to see that targeting and in vivo efficacy were retained,” she told GEN.

The researchers’ miRNA delivery technology was founded on work by colleague and co-author Philip Low, Ph.D., which indicated that folate conjugates in general are specifically taken up by tumor cells quite rapidly, even in humans, Dr. Kasinski explained. “Dr. Low’s studies suggested that RNAs conjugated to folate would be exposed to serum nucleases for a relatively short period of time. Coupled with the need to reduce toxicity associated with current delivery vehicles and increase targeting efficiency, we decided to explore the high-risk idea of unprotected RNA delivery.”

Using folate to target its receptor on cells isn’t a new concept. Dr. Kasinski noted that Dr. Low’s folate conjugates are entering a Phase III clinical trial for image-guided surgical resection, while in their paper, the authors state that “successful folate-targeted delivery, with payloads as diverse as small radiopharmaceutical agents to large DNA-containing formulations, has been exemplified at both the preclinical and clinical levels.”

However, they point out, folate-mediated delivery of small RNAs “lags behind,” because of the preconception that the RNA molecules must be protected from degradation in the circulation. And while attempts have been made to incorporate folate onto dendrimer, co-polymer, or liposomal carriers, the resulting complexes are large, which makes it harder for them to penetrate the dense extracellular matrix found in most solid tumors.

Just how miRNAs in the Purdue University team’s FolamiR conjugates are protected from degradation is something that the team can at present only speculate, Dr. Kasinski admitted to GEN. “Certainly the rapid uptake in vivo helps to reduce exposure of the conjugates to serum endonucleases. However, we provide in vitro evidence that the folate–miRNA conjugate is more stable than the miRNA mimic itself. Our current hypothesis is that folate protects one end from endonucleases and that this may explain at least some of the stability.”

miRNA therapeutics firm Mirna Therapeutics had previously been developing a miR-34 mimic MRX34 as its lead anticancer candidate, but a Phase I trial was halted in September last year due to multiple immune-related severe adverse events. The FDA subsequently imposed a full clinical hold, and all R&D activities were stopped during late 2016. Earlier this year, Mirna was merged into Synthetic Biotics™ firm, Synlogic.

There are a few differences between the Purdue team’s MiR-34a and Mirna Therapeutics’ MRX34, Dr. Kasinski stressed. “Mirna Therapeutics’ mimic included a completely unmodified mature strand; ours contains minimal modifications on the 3’ end with two 2'-O-methyl RNA bases. The passenger strand of the mimic used in MRX34 is unmodified expect for a C6-amine cap at the 5' end of the molecule. The cap is thought to prevent the passenger strand from acting like a guide/mature strand. Our passenger strand contains an RNA double modified with an azide linker on the 5' end (to attach to folate) and multiple (10/20) 2'-O-methyl RNA bases. The methylated bases we used will likely impart increased stability and prevent toxicity.”

What caused the toxic response to MRX34 is also not clear, Dr. Kasinski stated. “The vehicle and/or the RNA could be contributing to the toxic response. In our case, it is unlikely that folate will cause a toxic response, and due to specificity and rapid clearance of the FolamiRs, it is unlikely that the RNA would be toxic. Specificity of uptake allows for reduced dosing and rapid clearance of the conjugates reduces the time that that the RNA is in circulation. Both of these are advantageous for reducing potential toxicity.”

The Purdue University team is continuing its work with miRNA conjugates, GEN was informed. Major areas for research include the use of alternative ligand/receptor pairs to deliver miRNAs to tumors that don’t overexpress the folate receptor, and also mechanisms to promote endosomal release. “It is well known that folate conjugates are internalized via receptor-mediated endocytosis,” Dr. Kasinski commented. “This process results in the conjugates becoming trapped inside of endosomes. Our data suggest that at least some of the miRNA-34a is escaping the endosome; however, we envision even greater efficacy and reduced dosing if more of the miRNA can escape. We are working on second-generated conjugates to promote endosomal escape.”

The researchers envisage making future progress into clinical development “with the help of industrial colleagues.” The university has a provisional patent application filed and intends to file the full patent. “Upon doing so we would be very interested in working with a licensee.”

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