Gene Therapy Marches Forward

Illustration: Erica P. Johnson After years of methodically lumbering along with antisense and gene knockout technologies, gene therapy has been given fresh legs. Techniques such as RNA interference (RNAi)--small nuclear RNAs to mask aberrant splice sites--and transposon technologies that extend the lives of transgenes are offering more applications than previously thought possible. A trio of recent papers highlights these approaches to gene therapy. RNAi is being used to boost gene therapy efforts in treating Huntington disease (HD). The challenge of fighting autosomal dominant diseases such as HD lies in decreasing aberrant gene expression. Citing Thomas Tuschl's seminal work on RNAi as their inspiration,1 researchers at the University of Iowa, Iowa City, silenced gene expression in a group of dominantly inherited neruodegenerative diseases using RNAi in the animal tissue of a cell model. These diseases, known as polyglutamine expansion diseases,2 cause HD.3 "Each siRNA [small interfering RNA] sequence has to be tailored specifically to a gene sequence that would hybridize to the complementary DNA you're interested in inhibiting the translation of," says Beverly Davidson, the Roy J. Carver Chair in Internal Medicine at the University of Iowa. The 22-nucleotide-long, hairpin-shaped molecule of siRNA is tailor made for binding to mRNA. The siRNA recruits a protein complex that targets mRNA for degradation, prohibiting its normal production of proteins and reducing expression of stably expressed plasmids in cells, endogenous genes and transgenes in murine models. The ability to turn off certain genes proves promising in curing other diseases as well. The required application of the vector is dependent upon the gene targeted. "For a condition in which the diseased alleles are being continually expressed, you'd want siRNA to be from a vector that allows for long-term immunity of expression from that allele," says Davidson. While a brief exposure is required for genes that degrade once they are targeted, according to Davidson, the measuring stick of this gene therapy will be if these diseases can be reversed in addition to being muted. Davidson says she is currently testing the applicability of this approach to other adult-onset neurodegenerative diseases and is using it as a tool in other functional genomic experiments. THE OL' BAIT AND SWITCH A novel approach to treat thalassemia using gene therapy involves a lentivirus vector to correct rather than replace the genetic defect that causes several forms of hemoglobin disorders.4 Gene splicing machinery preferentially recognizes the incorrect splice sites, which interfere with the subsequent production of hemoglobin. Here, the defective RNA is masked with a molecule of small nuclear RNA called U7 snRNA, which is normally involved with processing mRNA for histones. According to Ryszard Kole, professor of pharmacology at the Lineberger Comprehensive Cancer Center at the University of North Carolina at Chapel Hill, U7 snRNA is modified to have a site complementary to the defective splice site on pre-mRNA. "This blocks the splice site, and when splicing machinery sees it can't use this site, it will go to the normal one," Kole says. Masking the defective RNA is a more effective means of repair than replacing the gene. A good copy of the gene produces excess RNA and hemoglobin, which can be detrimental as well. This technique should better facilitate the only known cure for thalassemias: bone marrow transplantation. Kole says the stem cells present in a patient's bone marrow could be injected with the virus and replanted in the patient to express this RNA without complications of compatibility. "And what's nice about the virus is that, in principal, you only need to use it once," Kole says. NOTHING LASTS FOREVER One-time use has also been the holy grail for the administration of adenoviruses, the long-time workhorses of gene therapy. While adenoviruses have been efficient in therapeutic gene expression, their limited shelf life has been an obstacle to researchers. "The transience of gene expression for genetic diseases is a huge disadvantage, but it's the nature of the beast," says Mark Kay, professor of pediatrics and genetics at Stanford University. "The readministration of these [adenovirus] vectors is a real problem, because an immune response to the vector code builds up so you can't deliver it more than once," Kay says. This problem may be eliminated soon. Kay and his colleagues have built an Adenovirus-transposon vector that stabilizes transgene expression in vivo for more than six months in murine models.5 The somatic integration of this delivery system was able to maintain therapeutic levels of human coagulation Factor IX in the mice for an extended time. "We showed the DNA was still stable even when the mouse liver cells were [artificially stimulated] to divide many more [times] than they ever would during a normal life span," Kay says. The transposon, obtained from a salmon species, possesses short sequences at both ends. If the protein transposases are made, it allows the DNA to be integrated into the host. "This is a way to transiently make transposase that will only recognize these specific ends and allow that DNA to integrate," Kay says. Like Kole, Kay points out that if applied to the correct cell type, this vector can be administered once for lifelong expression. Hal Cohen can be contacted at hcohen@the-scientist.com. References 1. S.M. Elbashir et al., "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature, 411:494-8, 2001. 2. H. Xia et al., "siRNA-mediated gene silencing in vitro and in vivo," Nature Biotechnology, 20:1006-10, October 2002. 3. R. Lewis, "Understanding Huntington's disease," The Scientist, 15[8]:14, April 16, 2001. 4. M.M. Vacek et al., "High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA," Blood, advance online publication, DOI:10.1182 /blood-2002-06-1869, Aug. 15, 2002. 5. S.R. Yant et al., "Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo," Nature Biotechnology, 20:999-1005, October 2002.

Gene Therapy Marches Forward

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