One giant genomic leap for gene transfer technology
Authors: Mathias Ackermann, Cornel Fraefel
Journal: Molecular Therapy (2003)

As he descended from the Eagle lunar module and set foot on the Moon's surface on July 20, 1969, Neil Armstrong proclaimed, “…one small step for man, one giant leap for mankind.” In this issue of Molecular Therapy, Wade-Martins and colleagues report their own potential “giant leap”—in their case for gene transfer technology to treat genetic disease [1Wade-Martins R. et al.Infectious delivery of a 135 kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells.Mol. Ther. 2003; (this issue.)PubMed Google Scholar]. As of March 19, 2003, 10,617 different loci of possible disease-causing variations in the human genome have been recorded in the Online Mendelian Inheritance in Man (OMIM) database [2Online Mendelian Inheritance in Man.http://www.ncbi.nlm.nih.gov/omim/Google Scholar]. Direct correction of genetic defects is certainly the most elegant and fundamental approach to the treatment of genetic disease, and viruses are attractive vectors for the delivery of genetic information into eukaryotic cells. Although the delivery of transgenes by viral vectors is fairly efficient, there remain many obstacles to their use, such as the appropriate control of transgene expression and the generation of adverse effects of the virus on either the target cell population or the organism harboring the treated cell. Although the cytotoxicity of the virus vector can be at least partly overcome by eliminating viral determinants of pathogenesis, stable and controlled expression of the transgene at physiological levels requires additional regulatory sequences, often both up and downstream of the transgene. However, the inclusion of additional genetic elements into a viral vector takes up precious and often quite limited space. The transgene capacity of different viral vectors varies considerably, ranging from 4500 bases in adeno-associated virus (AAV) to > 25,000 bp in poxvirus vectors. However, vector capacities as large as 150,000 bp have become accessible through the establishment of helper virus-free herpes simplex virus type 1 (HSV-1)-based amplicon vectors, in which nearly the entire viral genome can be replaced by the transgene of choice [3Spaete R.R. Frenkel N. The herpes simplex virus amplicon: a new eukaryotic defective-virus cloning-amplifying vector.Cell. 1982; 30: 295-304Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 4Fraefel C. et al.Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.J. Virol. 1996; 70: 7190-7197Crossref PubMed Google Scholar, 5Saeki Y. et al.Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors.Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (191) Google Scholar]. Recently, Wade-Martins and colleagues reported a method to generate HSV-1 amplicon vectors (iBACs) from a library of bacterial artificial chromosomes (BACs) covering the entire human genome [6Wade-Martins R. et al.An infectious transfer and expression system for genomic DNA loci in human and mouse cells.Nat. Biotechnol. 2001; 19: 1067-1070Crossref PubMed Scopus (158) Google Scholar]. In their new work, the authors make use of this system to provide evidence in vitro for stable and physiologically satisfactory complementation of a genetic defect in human cells. Familial hypercholesterolemia (FH) is caused by loss of function of the low-density lipoprotein receptor (LDLR). Previous attempts to correct models of this genetic disease failed due to poor expression of the LDLR transgene [7Chen S.J. et al.Prolonged correction of hyperlipidemia in mice with familial hypercholesterolemia using an adeno-associated viral vector expressing very-lowdensity lipoprotein receptor.Mol. Ther. 2000; 2: 256-261Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Rader D.J. Gene therapy for familial hypercholesterolemia.Nutr. Metab. Card. Dis. 2001; 11: 40-44PubMed Google Scholar]. Although the average human gene encompasses 3000 bp— well within the capacity of many viral vectors—the human LDLR locus covers at least 45,000 bp, exceeding the capacity of all viral vectors with the exception of HSV-1 amplicons and their derivatives. In fact, the large genetic capacity of the HSV-1 vectors allowed the authors to include substantial portions of potential regulatory sequences upstream and downstream of the locus—an important factor that may have been crucial in achieving physiological amounts of LDLR expression. Furthermore, the vector was equipped with genetic elements from the Epstein-Barr Virus (EBNA-1) and the latent origin of DNA replication (orip), which together are necessary and sufficient to confer genetic stability to the transgene and support its segregation to daughter cells upon cell division. Most excitingly, the iBACs expressed functional LDLR protein at appropriate levels that could be controlled through negative feedback imparted by cholesterol import into the transduced cells. Nevertheless, there are limitations to this impressive system. For example, the largest human gene encompasses 2,400,000 bp,> 10 times greater than the capacity of the HSV-1-based vector. However, other viruses may provide solutions to this problem: Some begomoviruses package the two components of their genomes into separate particles. Using this principle, giant transgenes may be divided and packaged into separate particles and, following delivery into the cell, reconstituted by homologous recombination. So, how does the advance in gene transfer reported by Wade-Martins and colleagues compare to the achievement of July 20, 1969? The distance from the Earth to the Moon is 384,187 km, but the distance from earth to the farthest orbit of Pluto is more than 20,000 times greater (52 astro-nomic units). The insert of Wade-Martin's iBAC is 135,000 bp in size, ∼ 20,000 times shorter than the entire human genome. Though perhaps not a “giant leap” for mankind, the new study certainly represents one giant genomic leap forward that should ultimately advance gene transfer strategies to treat human disease.