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New Method to Repair Faulty Genes Stirs Interest in Chimeraplasty Technique

New Method to Repair Faulty Genes Stirs Interest in Chimeraplasty Technique Scientists have developed a provocative new technique for correcting tiny but potentially devastating errors in mutant genes, an approach they hope will someday provide a strategy for treating at least some genetic disorders. The approach, known as chimeraplasty or targeted gene correction, is still in its infancy and faces many technical challenges before it is clear whether the method can move from bench to bedside. Moreover, some skeptics say that a number of investigators have had difficulty getting the procedure to work and question whether the approach will be generally applicable to a variety of cell types. But a growing number of laboratories are beginning to report encouraging results in experimental systems. Some investigators are enthusiastic about the technology because of its potential advantages over more conventional gene therapy approaches, which have largely sought to treat a genetic defect by using viruses or other vectors to introduce a correct copy of the gene into human cells. Virus-free gene therapy? "This new approach has many advantages over using viral vectors," said R. Michael Blaese, MD, of the National Human Genome Research Institute. During the past few years, Blaese, a pioneer in gene therapy research, has worked with chimeraplasty technology in his own laboratory and become a believer in its potential—so much so, that he recently became the chief scientific officer of the Pennsylvania biotechnology company that is commercializing chimeraplasty. The key element of the new technology is a synthetic paperclip-shaped oligonucleotide, or "oligo," a short stretch of nucleic acid that contains DNA interspersed with small amounts of RNA. Researchers design a chimeric RNA-DNA structure to seek out and pair with a specific mutant gene sequence and then trigger the cell's natural DNA repair machinery. (The term "chimeric" is used to reflect the fact the molecule consists of different kinds of genetic material.) This system, which normally identifies and repairs DNA errors that crop up, repairs the error in the mutated gene to match the correct version on the chimeric molecule. In contrast, traditional gene therapy requires the delivery of a huge amount of genetic material, often to correct a very small glitch in a gene. And because viruses and other vectors aren't large enough to ferry a whole gene, researchers attempt to deliver only a copy of the coding region. This compromise means that important DNA sequences that regulate the expression of the gene and coordinate the expression of other genes that may be linked to it are not included, said Blaese. Another drawback of viral vectors is that they integrate at random locations in the chromosomes—and being in different locations can dramatically influence the amount of gene expression. In some diseases, such as adenosine deaminase (ADA) deficiency, it may not matter how much ADA the transplanted gene makes, said Blaese. "But if you make too much insulin, for example, it could kill the patient." The chief appeal of chimera-directed gene repair is that it targets point mutations—a deletion, insertion, or "misspelling" of a single base pair—and functions as a molecular word processor to search out and replace the genetic typo. The rest of the gene, along with the genetic sequences that regulate its function, remains intact. The approach [of chimera-directed gene repair] may also be more likely than traditional gene therapy to succeed in treating genetic disorders caused by an autosomal dominant mutation. In disorders where one flawed copy of a gene is sufficient to cause disease, adding a second normal copy to the cell may not be enough to overcome the effects of the mutation, points out C. I. Edvard Smith, MD, PhD, whose laboratory at the Karolinska Institute in Huddinge,Sweden, is studying the technique. Targeting and correcting the flaw with a chimeric molecule may provide a better strategy for tackling such a disorder. Results in black and white In a study published last month (Nat Biotechnol. 1998;16:1343-1346), researchers from Jefferson Medical College in Philadelphia presented an elegantly visual demonstration of the ability of chimeraplasty to repair the gene mutation responsible for albinism in a strain of mice and restore that gene's normal function, allowing formerly albino cells to churn out black pigment. View LargeDownload Cells from albino mice have a genetic mutation that prevents them from making an enzyme (tyrosinase) necessary to produce the black pigment melanin (left), but descendants of an albino cell with a tyrosinase gene successfully repaired by chimeraplasty are readily able to make melanin (right). Photo credit: Vitali Alexeev, PhD, and Kyonggeun Yoon, PhD The targeted cells had a single point mutation in the gene for tyrosinase, which is necessary for the production of melanin and pigmentation. The investigators, Kyonggeun Yoon, PhD, and Vitali Alexeev, PhD, used a chimeric molecule capable of recognizing and replacing the mutated base responsible for the albino trait. Once the mutation was corrected, the albino cells regained the ability to make tyrosinase and produce melanin, a change made evident by cells acquiring the black color of the pigment. The investigators cloned the pigmented cells, and analysis showed that the genetic repair and the ability to produce melanin were both permanent and inherited by daughter cells, said Yoon, who developed the gene-targeting technique with Eric Kmiec, PhD, also of Jefferson Medical College and Kimeragen, Inc, of Newtown, Pa, a company he founded to commercialize the technology. But much more research is needed to understand the mechanisms involved in the new gene repair method, to optimize the design of such molecules, and to make the technology generally applicable, said Yoon. For example, Yoon said, it's not clear why the efficiency of the procedure, using the same cells and the same chimeric molecule, varies so widely. And no one really understands the mechanism that allows the RNA-DNA chimeric molecule to rewrite a genetic mistake. While investigators generally agree that the hybrid molecule stimulates the cell's DNA repair system, other factors are likely to play a role, such as the cell's machinery for recombination, she said. Because factors such as recombination frequency, cell cycle, and other elements vary among cell types, it may not be possible to apply the technology to all cells, she said. In other work, presented at the Chicago meeting in November of the American Association for the Study of Liver Diseases, researchers from the University of Minnesota Medical School, Minneapolis, and Albert Einstein College of Medicine, Bronx, NY, reported that they had succeeded in using chimeraplasty to correct a gene defect in a rat model for a human liver disease, Crigler-Najjar syndrome type I. The defective gene results in an enzyme deficiency that causes an accumulation of toxic levels of bilirubin. In the study, funded in part by Kimeragen, the researchers injected the chimeric molecules into the tail veins of 12 rats and injected a placebo into another dozen animals. Liver biopsies revealed that about 15% to 20% of the genes were corrected in samples from the treated animals, reducing bilirubin levels as much as 50% from pretreatment levels, said Betsy Kren, PhD. The investigators hope to begin testing the approach in human patients with Crigler-Najjar syndrome, perhaps by the end of 1999. Not only does the technology offer great potential for treating liver diseases, but the method also may be a valuable research tool, allowing researchers to "knock out" genes in adult tissues and cell types and gain insights into the function of the missing gene's product. Although the technology can bring about only small corrections—up to three consecutive base pairs in a gene—some researchers hope its applications may extend beyond repairing diseases caused by point mutations. It may be possible to use the technology to partially correct genetic errors more substantial than a point mutation, such as a deletion of a chunk of a gene, so that patients would develop a milder and less disabling form of the disease, said Thomas Rando, MD, PhD, of Stanford University School of Medicine. In males with a severe form of Duchenne muscular dystrophy, for example, a portion of the gene may be deleted in such a way that the protein it normally encodes, called dystrophin, is markedly reduced or absent in muscle cells. But if chimeraplasty could restore a cell's ability to "read" the code and make a flawed but semifunctional protein, the result could be a reduction in morbidity and a significantly expanded lifespan, as is the case in the milder Becker muscular dystrophy. But even if the chimeric molecule can successfully transform a severely crippled gene into one that will result a milder form of disease, finding a systemic delivery system to get the molecule into all relevant muscle cells, including the heart and diaphragm, will be a major barrier to overcome, said Rando. Proponents are also exploring other potential applications of chimera-directed gene repair. Researchers have found that the technique works when applied to plant and bacterial cells, pointing to possible applications in plant genetics and infectious disease. Another possibility is the creation of animals whose genetic makeup is modified so that their organs could be transplanted into humans without triggering acute rejection. Limitations? While a number of investigators are excited about chimeraplasty's potential, others are more cautious, saying that many laboratories have been unsuccessful in getting the technique to work or have achieved only modest rates of gene conversion. Some groups, however, say that using chimeric molecules of high quality and purity, coupled with an efficient means of homing in on the target cells, can result in gene repair rates high enough to have a dramatic clinical effect. The University of Minnesota researchers have achieved a gene conversion rate as high as 40% in one recently published animal study (Nat Med. 1998;4:285-290). In this "proof-of-principle" effort, the investigators introduced a mutation in the gene for blood clotting factor IX (causing hemophilia B) in rat liver cells, both in vivo and in vitro. The treatment resulted in a 50% reduction in coagulant activity, and the altered genes and their effects persisted as the rats aged, indicating a permanent change in their genetic makeup, said Clifford J. Steer, MD, who heads the group. Studies aimed at targeting and repairing a flawed factor IX gene in a strain of dogs with hemophilia B are under way. Another potential concern is the possibility of creating an unwanted "bystander effect" in normal genes, noted Smith. Of particular concern are closely related genes that belong to families. Such families include many different genes, such as those encoding structural proteins and signaling molecules. "Because large parts of these genes are nearly or completely identical, it is possible that a chimeric molecule aimed at correcting one such gene could accidently affect related genes," Smith cautioned. Perhaps the major hurdle for the technology is finding ways to efficiently deliver the molecule to appropriate cells or tissues, using vehicles such as liposomes or synthetic polymers. One reason the Minnesota group achieved such high rates of gene conversion relates to their delivery vehicle, explained Steer. They packaged the hybrid molecule in a protective polymer tagged with a sugar that is recognized by a receptor on liver cells. Although Blaese acknowledges that delivery is an important concern, he believes that finding ways to deliver small chimeric molecules will be easier than dealing with unwieldy whole-gene vectors—and that the rewards are likely to be greater. "If you're trying to treat genetic disease, you need finer tools than we've had with traditional gene therapy," said Blaese. "That's what I think chimeraplasty provides—a quantum leap forward in the kind of technology we have to work with and what we'll be able to do therapeutically." http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA American Medical Association

New Method to Repair Faulty Genes Stirs Interest in Chimeraplasty Technique

Stephenson, Joan
JAMA , Volume 281 (2) – Jan 13, 1999
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Publisher
American Medical Association
Copyright
Copyright © 1999 American Medical Association. All Rights Reserved.
ISSN
0098-7484
eISSN
1538-3598
DOI
10.1001/jama.281.2.119
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Abstract

Scientists have developed a provocative new technique for correcting tiny but potentially devastating errors in mutant genes, an approach they hope will someday provide a strategy for treating at least some genetic disorders. The approach, known as chimeraplasty or targeted gene correction, is still in its infancy and faces many technical challenges before it is clear whether the method can move from bench to bedside. Moreover, some skeptics say that a number of investigators have had difficulty getting the procedure to work and question whether the approach will be generally applicable to a variety of cell types. But a growing number of laboratories are beginning to report encouraging results in experimental systems. Some investigators are enthusiastic about the technology because of its potential advantages over more conventional gene therapy approaches, which have largely sought to treat a genetic defect by using viruses or other vectors to introduce a correct copy of the gene into human cells. Virus-free gene therapy? "This new approach has many advantages over using viral vectors," said R. Michael Blaese, MD, of the National Human Genome Research Institute. During the past few years, Blaese, a pioneer in gene therapy research, has worked with chimeraplasty technology in his own laboratory and become a believer in its potential—so much so, that he recently became the chief scientific officer of the Pennsylvania biotechnology company that is commercializing chimeraplasty. The key element of the new technology is a synthetic paperclip-shaped oligonucleotide, or "oligo," a short stretch of nucleic acid that contains DNA interspersed with small amounts of RNA. Researchers design a chimeric RNA-DNA structure to seek out and pair with a specific mutant gene sequence and then trigger the cell's natural DNA repair machinery. (The term "chimeric" is used to reflect the fact the molecule consists of different kinds of genetic material.) This system, which normally identifies and repairs DNA errors that crop up, repairs the error in the mutated gene to match the correct version on the chimeric molecule. In contrast, traditional gene therapy requires the delivery of a huge amount of genetic material, often to correct a very small glitch in a gene. And because viruses and other vectors aren't large enough to ferry a whole gene, researchers attempt to deliver only a copy of the coding region. This compromise means that important DNA sequences that regulate the expression of the gene and coordinate the expression of other genes that may be linked to it are not included, said Blaese. Another drawback of viral vectors is that they integrate at random locations in the chromosomes—and being in different locations can dramatically influence the amount of gene expression. In some diseases, such as adenosine deaminase (ADA) deficiency, it may not matter how much ADA the transplanted gene makes, said Blaese. "But if you make too much insulin, for example, it could kill the patient." The chief appeal of chimera-directed gene repair is that it targets point mutations—a deletion, insertion, or "misspelling" of a single base pair—and functions as a molecular word processor to search out and replace the genetic typo. The rest of the gene, along with the genetic sequences that regulate its function, remains intact. The approach [of chimera-directed gene repair] may also be more likely than traditional gene therapy to succeed in treating genetic disorders caused by an autosomal dominant mutation. In disorders where one flawed copy of a gene is sufficient to cause disease, adding a second normal copy to the cell may not be enough to overcome the effects of the mutation, points out C. I. Edvard Smith, MD, PhD, whose laboratory at the Karolinska Institute in Huddinge,Sweden, is studying the technique. Targeting and correcting the flaw with a chimeric molecule may provide a better strategy for tackling such a disorder. Results in black and white In a study published last month (Nat Biotechnol. 1998;16:1343-1346), researchers from Jefferson Medical College in Philadelphia presented an elegantly visual demonstration of the ability of chimeraplasty to repair the gene mutation responsible for albinism in a strain of mice and restore that gene's normal function, allowing formerly albino cells to churn out black pigment. View LargeDownload Cells from albino mice have a genetic mutation that prevents them from making an enzyme (tyrosinase) necessary to produce the black pigment melanin (left), but descendants of an albino cell with a tyrosinase gene successfully repaired by chimeraplasty are readily able to make melanin (right). Photo credit: Vitali Alexeev, PhD, and Kyonggeun Yoon, PhD The targeted cells had a single point mutation in the gene for tyrosinase, which is necessary for the production of melanin and pigmentation. The investigators, Kyonggeun Yoon, PhD, and Vitali Alexeev, PhD, used a chimeric molecule capable of recognizing and replacing the mutated base responsible for the albino trait. Once the mutation was corrected, the albino cells regained the ability to make tyrosinase and produce melanin, a change made evident by cells acquiring the black color of the pigment. The investigators cloned the pigmented cells, and analysis showed that the genetic repair and the ability to produce melanin were both permanent and inherited by daughter cells, said Yoon, who developed the gene-targeting technique with Eric Kmiec, PhD, also of Jefferson Medical College and Kimeragen, Inc, of Newtown, Pa, a company he founded to commercialize the technology. But much more research is needed to understand the mechanisms involved in the new gene repair method, to optimize the design of such molecules, and to make the technology generally applicable, said Yoon. For example, Yoon said, it's not clear why the efficiency of the procedure, using the same cells and the same chimeric molecule, varies so widely. And no one really understands the mechanism that allows the RNA-DNA chimeric molecule to rewrite a genetic mistake. While investigators generally agree that the hybrid molecule stimulates the cell's DNA repair system, other factors are likely to play a role, such as the cell's machinery for recombination, she said. Because factors such as recombination frequency, cell cycle, and other elements vary among cell types, it may not be possible to apply the technology to all cells, she said. In other work, presented at the Chicago meeting in November of the American Association for the Study of Liver Diseases, researchers from the University of Minnesota Medical School, Minneapolis, and Albert Einstein College of Medicine, Bronx, NY, reported that they had succeeded in using chimeraplasty to correct a gene defect in a rat model for a human liver disease, Crigler-Najjar syndrome type I. The defective gene results in an enzyme deficiency that causes an accumulation of toxic levels of bilirubin. In the study, funded in part by Kimeragen, the researchers injected the chimeric molecules into the tail veins of 12 rats and injected a placebo into another dozen animals. Liver biopsies revealed that about 15% to 20% of the genes were corrected in samples from the treated animals, reducing bilirubin levels as much as 50% from pretreatment levels, said Betsy Kren, PhD. The investigators hope to begin testing the approach in human patients with Crigler-Najjar syndrome, perhaps by the end of 1999. Not only does the technology offer great potential for treating liver diseases, but the method also may be a valuable research tool, allowing researchers to "knock out" genes in adult tissues and cell types and gain insights into the function of the missing gene's product. Although the technology can bring about only small corrections—up to three consecutive base pairs in a gene—some researchers hope its applications may extend beyond repairing diseases caused by point mutations. It may be possible to use the technology to partially correct genetic errors more substantial than a point mutation, such as a deletion of a chunk of a gene, so that patients would develop a milder and less disabling form of the disease, said Thomas Rando, MD, PhD, of Stanford University School of Medicine. In males with a severe form of Duchenne muscular dystrophy, for example, a portion of the gene may be deleted in such a way that the protein it normally encodes, called dystrophin, is markedly reduced or absent in muscle cells. But if chimeraplasty could restore a cell's ability to "read" the code and make a flawed but semifunctional protein, the result could be a reduction in morbidity and a significantly expanded lifespan, as is the case in the milder Becker muscular dystrophy. But even if the chimeric molecule can successfully transform a severely crippled gene into one that will result a milder form of disease, finding a systemic delivery system to get the molecule into all relevant muscle cells, including the heart and diaphragm, will be a major barrier to overcome, said Rando. Proponents are also exploring other potential applications of chimera-directed gene repair. Researchers have found that the technique works when applied to plant and bacterial cells, pointing to possible applications in plant genetics and infectious disease. Another possibility is the creation of animals whose genetic makeup is modified so that their organs could be transplanted into humans without triggering acute rejection. Limitations? While a number of investigators are excited about chimeraplasty's potential, others are more cautious, saying that many laboratories have been unsuccessful in getting the technique to work or have achieved only modest rates of gene conversion. Some groups, however, say that using chimeric molecules of high quality and purity, coupled with an efficient means of homing in on the target cells, can result in gene repair rates high enough to have a dramatic clinical effect. The University of Minnesota researchers have achieved a gene conversion rate as high as 40% in one recently published animal study (Nat Med. 1998;4:285-290). In this "proof-of-principle" effort, the investigators introduced a mutation in the gene for blood clotting factor IX (causing hemophilia B) in rat liver cells, both in vivo and in vitro. The treatment resulted in a 50% reduction in coagulant activity, and the altered genes and their effects persisted as the rats aged, indicating a permanent change in their genetic makeup, said Clifford J. Steer, MD, who heads the group. Studies aimed at targeting and repairing a flawed factor IX gene in a strain of dogs with hemophilia B are under way. Another potential concern is the possibility of creating an unwanted "bystander effect" in normal genes, noted Smith. Of particular concern are closely related genes that belong to families. Such families include many different genes, such as those encoding structural proteins and signaling molecules. "Because large parts of these genes are nearly or completely identical, it is possible that a chimeric molecule aimed at correcting one such gene could accidently affect related genes," Smith cautioned. Perhaps the major hurdle for the technology is finding ways to efficiently deliver the molecule to appropriate cells or tissues, using vehicles such as liposomes or synthetic polymers. One reason the Minnesota group achieved such high rates of gene conversion relates to their delivery vehicle, explained Steer. They packaged the hybrid molecule in a protective polymer tagged with a sugar that is recognized by a receptor on liver cells. Although Blaese acknowledges that delivery is an important concern, he believes that finding ways to deliver small chimeric molecules will be easier than dealing with unwieldy whole-gene vectors—and that the rewards are likely to be greater. "If you're trying to treat genetic disease, you need finer tools than we've had with traditional gene therapy," said Blaese. "That's what I think chimeraplasty provides—a quantum leap forward in the kind of technology we have to work with and what we'll be able to do therapeutically."

Journal

JAMA – American Medical Association

Published: Jan 13, 1999

Keywords: genes,transplantation chimera

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