Nanoparticles—The Next Big Thing?
Authors: Gina Kirchweger
Journal: Molecular Therapy (2002)

Latecomers onto the gene therapy scene, nanoparticles were long overshadowed by their viral counterparts. After all, modified viruses have been the best available tool to shuttle DNA into cells to make up for faulty genes. Over 70% of ongoing clinical trials in Europe and the United States employ viral vectors.However, since gene therapy patient Jesse Gelsinger died of a lethal immune reaction after the systemic delivery of an adenovirus, renewed concern over their safety spurred many scientists to take a closer look at nonviral approaches. “The problem with viral systems is that they can cause immune reactions, which makes safety a big issue,” says Robert Langer, professor of chemical and biomedical engineering at the Massachusetts Institute of Technology.Nonviral methods of gene delivery, including naked DNA and DNA condensed with agents, such as cationic lipids or polymers, were long considered less efficient. But as researchers learn how to boost the efficiency of the tiny, tightly packed DNA particles and fine-tune their properties, nanoparticles are catching up with viral vectors.Completely synthetic, nanoparticles can be designed to diminish an adverse immune response, and their production can be easily scaled up for pharmaceutical purposes. But they pose their own challenges. “The big issue with nonviral systems is to make them effective enough to do a good job,” adds Langer. Drawing from his extensive experience with controlled drug release he is trying to do just that. “One way to do it is to come up with a polymer that behaves exactly like a virus. You might be smart or get lucky. The other way is to increase your chances of luck by making not one or two but hundreds or thousands [of synthetic polymers]. That greatly increases your chances of having a hit,” he says.And that's what he opted for. In a parallel approach, he developed a method to synthesize large libraries of biodegradable cationic polymers and a high-throughput screening assay to identify new synthetic vector families with the required features: the polymers have to be able to condense or package DNA to small sizes so that it can be taken up by cells, stabilize DNA before and after cellular uptake, bypass or escape the cell's endocytic pathways, deliver the DNA to the cell's nucleus, and unpackage DNA in an active form [1Lynn D.M. Accelerated discovery of synthetic transfection vectors; parallel synthesis and screening of a degradable polymer library.J. Am. Chem. Soc. 2001; 123: 8155-8156Crossref PubMed Scopus (358) Google Scholar]. Some of the discovered packaging materials already showed encouraging results in animal models in which they were used to deliver vaccines and genes to brain tumors. “But ultimately it is not so much a nanoparticle issue but a much broader one. Part of the future is certainly having good enough targeting moieties available,” he notes and adds that “as we learn more and more [about the underlying biology] the bars for successful gene delivery will be lowered.”But in those instances when technology and biology are ready to join forces, nanoparticles can be fashioned into perfect messengers, delivering their genetic payload with precision. In an elegant approach, David Cheresh, a vascular biologist at the Scripps Research Institute in La Jolla, California, and colleagues recently demonstrated what the future might look like. He successfully harnessed the powers of nanoparticles to specifically target angiogenic blood vessels in mice and choke off the blood supply of tumors without influencing the normal blood vessels or any other tissues (Fig. 1).In cancer-related angiogenesis, tumors coax endothelial cells in surrounding blood vessels to proliferate and sprout new branches to secure their nourishment. Cancer researchers have long focused on a strategy of fighting cancer by interrupting these blood supply lines. But many anti-angiogenic therapies had setbacks. “Most of the current therapies that are directed against blood vessels are really not selective for angiogenic vessels. Toxicity issues that might be associated with gene therapy or any anti-angiogenic therapy are often associated with impacting other cells than the cells of interest,” explains Cheresh. “We felt that we could selectively manipulate those vessels, turn them off or kill them,” he says.One of the discoveries that made the novel approach possible was made many years ago in Cheresh's laboratory. They found that αvβ3, a member of the family of membrane proteins called integrins, is highly expressed on angiogenic blood vessels but not, or at least not in detectable levels, on established ones. In addition to serving as an unmistakable signpost, αvβ3 belongs to a family of receptors that adenoviruses and others use to gain entry into the cell to express their genes. After establishing that a small organic αvβ3 ligand coupled to nanoparticles could selectively deliver genes to αvβ3-bearing cells, one question remained: Which gene to pick?Tumors can produce up to 20 different growth factors that might elicit an angiogenic response. If you want to knock out a tumor you need to target more than one. “Many of the angiogenic therapies that are tested today in clinical trials only turn off one pathway and leave the others intact. We thought, let's go downstream and hit a factor that is central to all players,” remembers Cheresh. RAF1 kinase emerged as the ideal candidate; all pathways seemed to converge there.With the help of Mark Bednarski, an organic chemist and radiologist at Stanford University, Cheresh's team packed a mutant form of RAF1 that inhibits normal RAF1 activity into cationic lipid-based nanoparticles decorated with αvβ3 ligand. Then they injected these particles into the tail veins of mice carrying melanomas. Within 6 days of a single treatment the targeted vehicle had homed in on local endothelial cells that were expanding to form new blood vessels and forced them to self-destruct. Cut off from their supply lines, tumors of 400 mm3—huge for murine standards—disappeared. RAF1 also eliminated metastatic lesions in lung or liver. A closer look revealed evidence of apoptosis in the targeted vessels, surrounded by concentric rings of apoptosis in the neighboring tumor tissue [2Hood J.D. Tumor regression by targeted gene delivery to the neovasculature.Science. 2002; 296: 2404-2407Crossref PubMed Scopus (784) Google Scholar].Savio Woo, director of the Institute for Gene Therapy at the Mount Sinai School of Medicine in New York, thinks the results are wonderful and marvels at the combination of biological knowledge and efficacy. “The most critical aspect is the design of the delivery vehicle, which has a very high level of specificity making this approach possible. The second important component is the choice of the mutant RAF1, which of course is based on the biology of endothelial cells,” he says.The next step, says Cheresh, is to refine the technique and possibly also use it to stimulate vessel growth. Within hours of a stroke or a heart attack αvβ3 appears on the blood vessels in the damaged area. Using the same principle, nanoparticles could be used to deliver RAF1 to encourage angiogenesis. The San Diego-based company TargeGen is currently developing the method to test it in clinical trials in the future.Clinical phase I trials in a dozen cystic fibrosis (CF) patients have just begun with a different kind of nanoparticle: ultra-tiny balls, where a single molecule of the cystic fibrosis transmembrane regulator (CFTR) gene is scrunched up into particles no more than 25 nm across. At around 100 nm in size most nanoparticles are too large to pass through the tiny pores in the nuclear membrane, which are only 25 to 50 nm wide. It is thought that DNA enters the nucleus during cell division because the membrane breaks down during mitosis. “Our data suggests that you need to be smaller than the internal diameter of the nuclear pore to be really effective. I think size is a characteristic that will be key for nonviral vectors,” says Mark Cooper, vice president of science and medical affairs at Copernicus Therapeutics in Cleveland, Ohio.With polylysine as their primary packing material, they can change the shape of the particle to obtain a minimal cross-sectional diameter, allowing the plasmid to slip into the nucleus [3Liu G. Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation.J. Biol. Chem. 2001; 276: 34379-34384Crossref PubMed Scopus (154) Google Scholar]. When Pam Davis of Case Western University School of Medicine, who pioneered this method, tried the technique on mice with CF, she found the replacement gene was expressed in nasal lining and partially restored function with little or no immune reaction [4Ziady, A. G., et al.2002. Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA complexes targeted to the serpin-enzyme complex receptor. Mol. Ther.5: 413–419,Google Scholar]. Describing unpublished results, Cooper adds that “when we transfect a mouse lung the transfer efficiency is very high, in some cases 70–80% of the airway cells. That means we are mainly transfecting nondividing cells.”But Michael Konstan, Director of the LeRoy Matthews Cystic Fibrosis Center, University Hospitals of Cleveland, who leads the clinical study, warns that pulmonary pathology in CF mice is not as pronounced as in humans. Nevertheless he is optimistic that nanoparticles might be the answer to the many failed attempts to treat CF with gene therapy. “I am on the clinical side and I have been waiting to apply some form of gene therapy experiment to our patients here for the past 10 years. We basically have refused to participate in any of the viral approaches because I didn't want to subject my patients to anything I am not convinced will be a therapy down the road,” says Konstan. He expects first results at the end of the year.Asked to predict the future of nanoparticles, Woo hesitates. “There is no such thing as a perfect vector. Both viral systems and systems based on nanoparticles will find major applications depending on the biology and pathophysiology of the disease in question,” he says. Latecomers onto the gene therapy scene, nanoparticles were long overshadowed by their viral counterparts. After all, modified viruses have been the best available tool to shuttle DNA into cells to make up for faulty genes. Over 70% of ongoing clinical trials in Europe and the United States employ viral vectors. However, since gene therapy patient Jesse Gelsinger died of a lethal immune reaction after the systemic delivery of an adenovirus, renewed concern over their safety spurred many scientists to take a closer look at nonviral approaches. “The problem with viral systems is that they can cause immune reactions, which makes safety a big issue,” says Robert Langer, professor of chemical and biomedical engineering at the Massachusetts Institute of Technology. Nonviral methods of gene delivery, including naked DNA and DNA condensed with agents, such as cationic lipids or polymers, were long considered less efficient. But as researchers learn how to boost the efficiency of the tiny, tightly packed DNA particles and fine-tune their properties, nanoparticles are catching up with viral vectors. Completely synthetic, nanoparticles can be designed to diminish an adverse immune response, and their production can be easily scaled up for pharmaceutical purposes. But they pose their own challenges. “The big issue with nonviral systems is to make them effective enough to do a good job,” adds Langer. Drawing from his extensive experience with controlled drug release he is trying to do just that. “One way to do it is to come up with a polymer that behaves exactly like a virus. You might be smart or get lucky. The other way is to increase your chances of luck by making not one or two but hundreds or thousands [of synthetic polymers]. That greatly increases your chances of having a hit,” he says. And that's what he opted for. In a parallel approach, he developed a method to synthesize large libraries of biodegradable cationic polymers and a high-throughput screening assay to identify new synthetic vector families with the required features: the polymers have to be able to condense or package DNA to small sizes so that it can be taken up by cells, stabilize DNA before and after cellular uptake, bypass or escape the cell's endocytic pathways, deliver the DNA to the cell's nucleus, and unpackage DNA in an active form [1Lynn D.M. Accelerated discovery of synthetic transfection vectors; parallel synthesis and screening of a degradable polymer library.J. Am. Chem. Soc. 2001; 123: 8155-8156Crossref PubMed Scopus (358) Google Scholar]. Some of the discovered packaging materials already showed encouraging results in animal models in which they were used to deliver vaccines and genes to brain tumors. “But ultimately it is not so much a nanoparticle issue but a much broader one. Part of the future is certainly having good enough targeting moieties available,” he notes and adds that “as we learn more and more [about the underlying biology] the bars for successful gene delivery will be lowered.” But in those instances when technology and biology are ready to join forces, nanoparticles can be fashioned into perfect messengers, delivering their genetic payload with precision. In an elegant approach, David Cheresh, a vascular biologist at the Scripps Research Institute in La Jolla, California, and colleagues recently demonstrated what the future might look like. He successfully harnessed the powers of nanoparticles to specifically target angiogenic blood vessels in mice and choke off the blood supply of tumors without influencing the normal blood vessels or any other tissues (Fig. 1). In cancer-related angiogenesis, tumors coax endothelial cells in surrounding blood vessels to proliferate and sprout new branches to secure their nourishment. Cancer researchers have long focused on a strategy of fighting cancer by interrupting these blood supply lines. But many anti-angiogenic therapies had setbacks. “Most of the current therapies that are directed against blood vessels are really not selective for angiogenic vessels. Toxicity issues that might be associated with gene therapy or any anti-angiogenic therapy are often associated with impacting other cells than the cells of interest,” explains Cheresh. “We felt that we could selectively manipulate those vessels, turn them off or kill them,” he says. One of the discoveries that made the novel approach possible was made many years ago in Cheresh's laboratory. They found that αvβ3, a member of the family of membrane proteins called integrins, is highly expressed on angiogenic blood vessels but not, or at least not in detectable levels, on established ones. In addition to serving as an unmistakable signpost, αvβ3 belongs to a family of receptors that adenoviruses and others use to gain entry into the cell to express their genes. After establishing that a small organic αvβ3 ligand coupled to nanoparticles could selectively deliver genes to αvβ3-bearing cells, one question remained: Which gene to pick? Tumors can produce up to 20 different growth factors that might elicit an angiogenic response. If you want to knock out a tumor you need to target more than one. “Many of the angiogenic therapies that are tested today in clinical trials only turn off one pathway and leave the others intact. We thought, let's go downstream and hit a factor that is central to all players,” remembers Cheresh. RAF1 kinase emerged as the ideal candidate; all pathways seemed to converge there. With the help of Mark Bednarski, an organic chemist and radiologist at Stanford University, Cheresh's team packed a mutant form of RAF1 that inhibits normal RAF1 activity into cationic lipid-based nanoparticles decorated with αvβ3 ligand. Then they injected these particles into the tail veins of mice carrying melanomas. Within 6 days of a single treatment the targeted vehicle had homed in on local endothelial cells that were expanding to form new blood vessels and forced them to self-destruct. Cut off from their supply lines, tumors of 400 mm3—huge for murine standards—disappeared. RAF1 also eliminated metastatic lesions in lung or liver. A closer look revealed evidence of apoptosis in the targeted vessels, surrounded by concentric rings of apoptosis in the neighboring tumor tissue [2Hood J.D. Tumor regression by targeted gene delivery to the neovasculature.Science. 2002; 296: 2404-2407Crossref PubMed Scopus (784) Google Scholar]. Savio Woo, director of the Institute for Gene Therapy at the Mount Sinai School of Medicine in New York, thinks the results are wonderful and marvels at the combination of biological knowledge and efficacy. “The most critical aspect is the design of the delivery vehicle, which has a very high level of specificity making this approach possible. The second important component is the choice of the mutant RAF1, which of course is based on the biology of endothelial cells,” he says. The next step, says Cheresh, is to refine the technique and possibly also use it to stimulate vessel growth. Within hours of a stroke or a heart attack αvβ3 appears on the blood vessels in the damaged area. Using the same principle, nanoparticles could be used to deliver RAF1 to encourage angiogenesis. The San Diego-based company TargeGen is currently developing the method to test it in clinical trials in the future. Clinical phase I trials in a dozen cystic fibrosis (CF) patients have just begun with a different kind of nanoparticle: ultra-tiny balls, where a single molecule of the cystic fibrosis transmembrane regulator (CFTR) gene is scrunched up into particles no more than 25 nm across. At around 100 nm in size most nanoparticles are too large to pass through the tiny pores in the nuclear membrane, which are only 25 to 50 nm wide. It is thought that DNA enters the nucleus during cell division because the membrane breaks down during mitosis. “Our data suggests that you need to be smaller than the internal diameter of the nuclear pore to be really effective. I think size is a characteristic that will be key for nonviral vectors,” says Mark Cooper, vice president of science and medical affairs at Copernicus Therapeutics in Cleveland, Ohio. With polylysine as their primary packing material, they can change the shape of the particle to obtain a minimal cross-sectional diameter, allowing the plasmid to slip into the nucleus [3Liu G. Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation.J. Biol. Chem. 2001; 276: 34379-34384Crossref PubMed Scopus (154) Google Scholar]. When Pam Davis of Case Western University School of Medicine, who pioneered this method, tried the technique on mice with CF, she found the replacement gene was expressed in nasal lining and partially restored function with little or no immune reaction [4Ziady, A. G., et al.2002. Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA complexes targeted to the serpin-enzyme complex receptor. Mol. Ther.5: 413–419,Google Scholar]. Describing unpublished results, Cooper adds that “when we transfect a mouse lung the transfer efficiency is very high, in some cases 70–80% of the airway cells. That means we are mainly transfecting nondividing cells.” But Michael Konstan, Director of the LeRoy Matthews Cystic Fibrosis Center, University Hospitals of Cleveland, who leads the clinical study, warns that pulmonary pathology in CF mice is not as pronounced as in humans. Nevertheless he is optimistic that nanoparticles might be the answer to the many failed attempts to treat CF with gene therapy. “I am on the clinical side and I have been waiting to apply some form of gene therapy experiment to our patients here for the past 10 years. We basically have refused to participate in any of the viral approaches because I didn't want to subject my patients to anything I am not convinced will be a therapy down the road,” says Konstan. He expects first results at the end of the year. Asked to predict the future of nanoparticles, Woo hesitates. “There is no such thing as a perfect vector. Both viral systems and systems based on nanoparticles will find major applications depending on the biology and pathophysiology of the disease in question,” he says.