Nanoparticles and Sirna – Partners on the Pathway to New Cancer

NCI Alliance for Nanotechnology in Cancer Monthly Feature August 2006

tility of nanoparticles gives us the ability to Nanoparticles and siRNA – better target cancer cells and to even develop nanoparticles that only deliver their RNA contents once they’ve entered the target cell. We hope that by taking advan- Partners on the Pathway tage of these opportunities, that we can head off potential problems.” to New Cancer Therapies At Caltech, for example, the Davis group has developed nanoparticles that provide effective release of their contents under the low pH conditions characteristic of intra- Seven short years ago, in an October cles, we have the ability to load large cellular environments experienced when 1999 paper published in the journal Science, numbers of these molecules into a protect- nanoparticles enter cells via targeted cancer the research community learned of a radi- ed environment, target them to cancer cell surface receptors. The resulting cally new mechanism that cells can use to cells, and then have them taken up effi- nanoparticles can carry significant control protein production. Discovered by ciently by those cells and release the RNA amounts of chemically unmodified siRNA plant geneticists David Baulcombe, Ph.D., molecules into the interior of those cells,” and effectively deliver them to targeted and Andrew Hamilton, Ph.D., this new says Mark Davis, Ph.D., at the California cells as his group showed in a paper pub- process relied on small pieces of double- Institute of Technology (Caltech). lished in Cancer Research last year. Davis, stranded RNA molecules that could bind to who is a founder of Calando and has and neutralize specific messenger RNA mol- In fact, the marriage between nanoparticles licensed his lab’s work to the company, ecules, which then prevented the cell from and siRNA is proving to be such a good says he hopes to have an anticancer siRNA translating that particular message into a one that several companies are already agent in clinical trials next year. protein. Then, in early 2002, a paper in pushing nanoparticle-delivered anticancer Nature from Thomas Tuschl, Ph.D., and his siRNA agents toward human clinical trials. Meanwhile, Rotello and his colleagues are collaborators at the Max-Planck Institute Intradigm, in Rockville, MD, expects to creating nanoparticles studded with mole- for Biophysical Chemistry in Gottingen, file an Investigational New Drug applica- cules of folic acid, a well-studied nanoparti- Germany, showed that small interfering tion with the U.S. Food and Drug cle targeting agent that binds to a receptor RNA (siRNA) molecules could block pro- Administration next year and begin clini- found in abundance on many types of can- tein production in mammalian cells. cal trials with its nanoparticulate formulation of an siRNA agent designed to stop Today, RNA silencing – the process trig- tumor-associated angiogenesis. And in What is an siRNA? gered by siRNA molecules – is poised to February of this year, Calando siRNAs have a well-defined structure: a short (usually have a major impact on the treatment of Pharmaceuticals, in Pasadena, CA, and the 21-nucleotide-long) double-strand of RNA (dsRNA) human ailments, particularly cancer. Using National Cancer Institute (NCI) entered with 2-nucleotide 3' overhangs on either end: siRNA molecules, researchers believe they into a collaborative development program can turn off the ability of cancer cells to for a nanoparticle-based siRNA therapeu- produce the key proteins that make them tic aimed at treating neuroblastoma, the different from normal cells, and by doing most common extracranial solid tumor in so, stop malignancy in its tracks. Early children younger than five years old. proof-of-principle experiments in various tumor cells showed quickly that RNA Targeting Is a Key silencing had great potential as a means for One characteristic of nanoparticles that Each strand has a 5' phosphate group and a 3' treating cancer. has made them a favorite among cancer hydroxyl (-OH) group. This structure is the result of researchers in general, and not just those processing by an enzyme that converts either long But almost as soon as those early results using siRNA, is the ability to decorate the dsRNAs or hairpin RNAs into siRNAs. siRNAs can also were in, a major obstacle surfaced: The surfaces of these drug vehicles with be introduced into cells using nanoparticles and human body is well-equipped to destroy tumor-targeting agents. But with siRNA, other methods to bring about the specific knock- double-stranded RNA circulating in blood this property is particularly attractive down of a gene of interest. Essentially any gene of and prevent it from entering cells. As a because there is some concern that siRNA which the sequence is known can thus be targeted result, getting these potential therapeutic delivered to the wrong cells may have based on sequence complementarity with an appro- agents to tumors and getting them into unintended consequences. “The siRNA priately tailored siRNA. This method has made siRNAs cancer cells was not going to be easy. field itself is so young that we just aren’t an important tool for gene function and drug target sure yet how important it is to target just Enter nanotechnology. Nanoparticles, it validation studies in the post-genomic era. one particular type of cell,” says Vincent Source: Wikipedia http://en.wikipedia.org/wiki/SiRNA turns out, are proving to be ideal carriers Rotello, Ph.D., of the University of for siRNA molecules. “With nanoparti- Massachusetts in Amherst. “But the versa-

NCI Alliance for Nanotechnology in Cancer1 Monthly Feature August 2006 NCI Alliance for Nanotechnology in Cancer M o n t h l y F e a t u r e August 2006

any nanoparticle that is to be used in the "The results are very promising, but we body – not too big, not too small." still have several hurdles to jump before we can test this therapy on people," Guo says. Particles larger than about 100 nanome- "First and foremost, we must ensure that it ters are generally too large to pass through is as safe as we think it is. Some RNA can cell membranes into the cell's interior, be toxic to non-cancerous cells as well, and Guo says, and the body has a hard time though our nanoparticles appear to go retaining particles smaller than 10 straight to the cancer cells, where we want nanometers. But the tiny triangles fit, and them to go, we have to be sure they do not they worked well enough to interrupt the go anywhere else before we can inject them growth of human breast cancer cells and into a living person." leukemia model lymphocytes in laborato- ry experiments. The use of such protein- Stability of the RNA also is a factor the free particles could avoid the induction of team must consider. Although his group immune response and avoid antibody pro- had previously published data indicating duction and cell immunity. Therefore, it that phi29 RNA nanoparticles are more facilitates the long-term treatment in stable than other RNA, Guo said the team A triangular nanoparticle made of RNA carriers chronic diseases such as cancer, hepatitis has also introduced chemical modifications uses both folic acid and a receptor-binding B, or AIDS. into the RNA backbone that further stabi- aptamer as tumor-targeting agents to deliver lizes it against degradation. anticancer siRNA molecules. "One characteristic of cancer cells is that Courtesy: Peixuan Guo, Purdue University they do not stop growing, which is one Intradigm’s approach to successfully deliver reason tumors develop," Guo says. "Once and target its lead anticancer siRNA agent inside, the siRNA essentially instructs the is to use a multicomponent polymer system cells to 'stop not stopping.' The nanoparti- that self-assembles into a rugged nanoparti- cer. But in addition, the surface of these cles had done their work on the breast can- cle when mixed with siRNA molecules. nanoparticles is covered with a sulfur-con- cer cell cultures within a few days." Martin Woodle, Ph.D., chief scientific offi- taining chemical group known as a thiol, cer at the company, leads the company’s with the sulfur bonding to the surface of Additionally, the team found that the efforts, which are aimed at developing an the gold nanoparticle. Cells contain large nanoparticles completely block cancer siRNA agent that silences the production amounts of the molecule glutathione, which development in living mice. A group of of a receptor for vascular endothelial is also a thiol. The abundance of glu- mice that was in the process of developing growth factor (VEGF) on the newly devel- tathione inside a cell results in the displace- cancer was tested with the nanoparticles, oping blood vessels surrounding tumors. ment of the thiols on the particle, triggering and they did not develop the disease. A sec- The idea behind this approach is that if the release of the RNA payloads. ond group, that was tested with mutated these blood vessels cannot respond to inactive RNA, all developed tumors. Peixuan Guo, Ph.D., at Purdue University’s VEGF, angiogenesis will stop and tumors Cancer Research Center, is taking advan- tage of another natural process to achieve targeted delivery and release of siRNA inside cancer cells. Nearly 20 years ago, Guo discovered that a particular bacteria- infecting virus called phi29 uses a novel type of RNA to package its DNA into its protein shell. Guo named his discovery pRNA, for packaging RNA, and today he and his colleagues are using it as a compo- nent of an all-RNA nanoparticle that resembles a triangle. Tests in both cultured human tumor cells and in mouse models of cancer have shown these RNA nanoparticles have potential as anticancer agents. Guo's team created their nanoparticles by linking together two different kinds of RNA: pRNA, which acts as the delivery vehicle, and siRNA, which acts as the therapeutic agent. The team then added folic acid molecules as the targeting agent. “Using techniques we learned from our earlier work,” explains Guo, “we were able Simplified schematic of the key steps required for siRNA delivery to and function within mammalian cells. to combine [these molecules] into triangles that are between 25 and 40 nanome- Courtesy: Derek Bartlett and Mark Davis, Nucleic Acids Research and Oxford University Press. Nucleic Acids ters wide. This is the Goldilocks size for Res. 2006; 34(1): 322–333. Published online 2006 January 12. doi: 10.1093/nar/gkj439

NCI Alliance for Nanotechnology in Cancer2 Monthly Feature August 2006 NCI Alliance for Nanotechnology in Cancer M o n t h l y F e a t u r e August 2006 will not be able to procure the nourish- ment they need to thrive and metastasize. Mice with Ewing’s sarcoma Four weeks after inoculation were treated with a targeted Woodle and his colleagues start with the nanoparticle containing anti- biocompatible polymer poly(ethyl- tumor siRNA (#31-40), as well eneimine), or PEI, and link it to a second as various control formula- biocompatible polymer, poly(ethylene gly- # 1-10 tions. Only the targeted agent col), also known as PEG. As a targeting had a marked effect on tumor agent, the researchers chose a three-amino- growth. Tumors show up as acid peptide, arginine-glycine-aspartic acid 10 purple areas. (known by its amino acid code RCG) that #11-20 Courtesy: Calando binds to a class of proteins known as inte- 8 grins. Found on the surface of new blood photon/sec/cm^2/sr Pharmaceuticals vessels, integrins are involved in a wide 6

range of biological processes, including x 10

angiogenesis, tumor cell growth and metas- 8 tasis, and inflammation. The researchers #21-30 4 attached this integrin-targeting peptide to one end of the PEG polymer chain. 2 Mixing equal parts of the polymer-RGD Color Bar construct and anti-VEGF receptor siRNA #31-40 Min = 1e-05 yields nanoparticles in which a core of Max = 1e-07 PEI and siRNA is surrounded by a water- soluble shell of PEG, with the RGD-targeting peptide sticking out from the particle. Studies in cultured cells showed that #41-50 this formulation entered only those cells containing integrins, suggesting that the RGD peptide was an effective targeting agent. In contrast, nanoparticles lacking RGD did not enter targeted cells. This study also showed that siRNA cargo was was having the desired effect – tumor RNA molecules is not trivial or inexpen- released within the cell and was able to growth in the animals treated with that for- sive, though chemists have made marked silence VEGF receptor production. mulation was minimal, compared to signif- improvements in reducing both the com- icant growth seen in both of the two con- plexity and cost of RNA production. Next, the Intradigm research team trol groups. Intradigm hopes to begin clini- administered their nanoparticle to tumor- cal trials with this agent late next year. “Obviously, all of these agents will have to bearing mice; they also treated a second prove themselves in clinical trials and until group of mice with a nanoparticle Based on these results and others, the they do, they’ll remain in the promising containing siRNA designed to silence a future of nanoparticle-based siRNA therapy but unproven category,” says Davis. “But bacterial protein (to act as a control experi- for cancer looks promising. Certainly, ques- combining siRNA with nanoparticle tech- ment) and left a third group of animals tions remain concerning the safety of these nology gives us a real opportunity to have untreated. Within six days of intravenous agents, though clinical trials using siRNA an impact in the treatment of cancer.” injection, it was clear that the nanoparticle to treat other illnesses have not yet come containing the anti-VEGF receptor siRNA across any stop signs. Also, manufacturing —Joe Alper

Selected References Devi RS. siRNA-based approaches in cancer therapy. Cancer Gene Therapy 13: 819-29, 2006. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411 (6836): 494-8, 2002. Guo S, Huang F, Guo P. Construction of a folate-conjugated pRNA of bacteriaphage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells. Gene Therapy 13: 814-20, 2006. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286 (5441): 950-2, 1999. Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, Triche TJ. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. Cancer Res. 65 (19): 8984-92, 2005. Khaled A, Guo S, Li F, Fuo P. Controllable self-assembly of nanoparticles for specific delivery of multiple therapeutic molecules to cancer cells using RNA nanotechnology. Nano Letters 5 (9): 1797-1808, 2005. Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, Molema G, Lu PY, Scaria PV, Woodle MC. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res. 32 (19): e1492004, 2004.

NCI Alliance for Nanotechnology in Cancer3 Monthly Feature August 2006