Gene Therapy (2005) 12, S18–S27 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt CONFERENCE PAPER Gutless adenovirus: last-generation adenovirus for gene therapy R Alba1, A Bosch1 and M Chillon1,2 1Gene Therapy Laboratory, Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy (CBATEG), Universitat Auto`noma de Barcelona, Bellaterra, Spain; and 2Institut Catala` de Recerca i Estudis Avanc¸ats (ICREA), Barcelona, Spain Last-generation adenovirus vectors, also called helper-depen- viral coding regions, gutless vectors require viral proteins dent or gutless adenovirus, are very attractive for gene therapy supplied in trans by a helper virus. To remove contamination because the associated in vivo immune response is highly by a helper virus from the final preparation, different systems reduced compared to first- and second-generation adenovirus based on the excision of the helper-packaging signal have vectors, while maintaining high transduction efficiency and been generated. Among them, Cre-loxP system is mostly tropism. Nowadays, gutless adenovirus is administered in used, although contamination levels still are 0.1–1% too high different organs, such as the liver, muscle or the central to be used in clinical trials. Recently developed strategies to nervous system achieving high-level and long-term transgene avoid/reduce helper contamination were reviewed. expression in rodents and primates. However, as devoid of all Gene Therapy (2005) 12, S18–S27. doi:10.1038/sj.gt.3302612 Keywords: adenovirus; gutless; helper-dependent vectors; in vivo gene therapy Introduction clinical for more information). Nowadays, adenovirus vectors are applied to treat cancer, monogenic disorders, Gene therapy for most genetic diseases requires expres- vascular diseases and others complications. sion of the therapeutic protein for the whole life of Basically, wild-type adenovirus is associated with the patient. In order to be efficient for the treatment mild diseases like conjunctivitis, pharyngitis and, in a of genetic disorders, a gene therapy vector has to meet high percentage, with colds or acute respiratory diseases several conditions: (i) safety, which can be better but not to tumoral pathways or other viral alterations.1 In achieved with a nonintegrative vector, as it avoids the the early 1950s, adenovirus was used for the first time risk for insertional mutagenesis; (ii) ability to be easily as a preventive vaccine for respiratory diseases. These and inexpensively produced at a large-scale in the studies supposed a great advance since they allowed the laboratory; (iii) stability in target cells, which is favored knowledge of aspects related to associated immune with low-immunogenic vectors; and (iv) high-capacity responses and adverse secondary effects. allowing the possibility of introducing full-length DNA As gene transfer vector, the adenovirus has a high sequences for most genes, long full-length cDNAs, transfection efficiency, both in quiescent and in dividing endogenous promoters or additional regulatory cells, it does not integrate; allows easy capsid modification sequences such as enhancers or insulators, which can in order to retarget its tropism to different tissues; and provide a tightly regulated expression of the therapeutic high titers (up to 1013 particles/ml) are routinely obtained. gene, similar to physiologic conditions. Adenoviruses In addition, it is noteworthy that in the last 10 years, new and especially gutless adenoviruses seem to accomplish scale-up adenovirus production systems have been most of these conditions as they are not integrative, they developed, facilitating its use in human clinical trials. have a capacity of 36 kb, they can be easily produced at Well-characterized human serotypes Ad2 and Ad5 high titers in the laboratory and can be delivered to a from group C are the classic adenoviruses used as large mass of cells, and, contrary to the first-generation vectors. In order to produce safe and nonreplicative adenovirus in which the expression of wild-type adeno- vectors, first-generation adenoviral vectors containing viral genes stimulates the immune system, gutless the whole viral genome with the exception of the E1 adenoviruses show long-term stability in many tissues. region were developed.2 To propagate first-generation As seen in Table 1, adenovirus is one of the first elections adenoviruses, several E1-expressing cell lines have been in clinical trials, being the most used vector in the last generated: 293,3 911,4 N52.E65 and PER.C6.6 5 years (1999–2004) (visit www.wiley.co.uk/genmed/ Although E1-deleted vectors cannot replicate in vivo, residual expression from adenoviral genes triggers a cytotoxic T lymphocyte (CTL) immune response towards Correspondence: Dr M Chillo´n, Gene Therapy Laboratory, Department of 7 Biochemistry and Molecular Biology, Center of Animal Biotechnology and infected cells, which finally leads to the elimination of Gene Therapy (CBATEG), Edifici H, Universitat Auto`noma de Barcelona, transduced cells and, therefore, to the lost of therapeutic Bellaterra 08193, Spain gene expression. Gutless adenovirus R Alba et al S19 To avoid this problem, second-generation adenoviral non-human DNA.14 However, first injections of gutless vectors combining deletion of different early regions vectors carrying lambda DNA elicited CTL immune (E17E3 and E2/E4) were generated (Figure 1). Deletion response because peptides from stuffer were presented of these regions permits to accommodate up to 14 kb and in the cell membrane, and it became evident that DNA hence increase the vector cloning capacity.8,9 However, stuffer played an important role in the stabilization of second-generation Ad vectors still do not avoid in vivo- viral DNA into the cell.15,16 The first choice was then associated immunogenicity and toxicity due to residual DNA from mammalian and human introns since they gene expression from remaining viral genes. seemed to favor maintenance of the gutless genome into Third-generation vectors, called gutless or gutted the cell for long periods of time.15 For example, intronic Ad, devoid of all coding viral regions were recently sequences from the HPRT gene containing matrix generated. They are also called helper-dependent adeno- attachment regions (MAR) elements have been used as viruses because of the need of a helper adenovirus that stuffer DNA showing increased DNA stability and no carries all coding regions, and high-capacity adenoviruses induction of a CTL response. Sequences from other because they can accommodate up to 36 kb of DNA. human loci have also been used with similar or better Briefly, the gutless adenovirus only keeps the 50 and 30 results.17 inverted terminal repeats (ITRs) and the packaging However, not all intronic sequences are appropriate signal (C) from the wild-type adenovirus. DNA stuffer and, thus, proper candidates should avoid Vector capsids package efficiently only 75–105% of the the following sequences (follow next characteristics) whole adenovirus genome.10–14 As therapeutic expres- (i) coding regions, (ii) repetitive sequences (like alu sion cassettes usually do not reach up to 36 kb, there is a sequences), (iii) hot spots for recombination, (iv) regions need to use stuffer DNA in order to complete the genome that can interfere with the expression of the transgene, size for encapsidation. Initially, it was believed that and (v) toxic or immunogenic regions. On the contrary, stuffer DNA’s unique role was the participation in the MAR that seem to stabilize the adenovirus genome into packaging of the vector genome. Thus, the first DNA the nucleus and permit long periods of gene expression stuffer used was from lambda phage, yeast, bacterial and are recommended.17 Table 1 Vectors used in gene therapy clinical trials Production of gutless adenovirus vectors Total number of Protocol number Since gutless vectors are devoid of all viral genes, protocols (2004) increase (1999–2004) proteins needed for its genome replication, packaging and capsid formation must be supplied in trans. This Retrovirus 263 83 is achieved by coinfection of the gutless with a helper Adenovirus 258 172 Lipofection 85 10 adenovirus. However, since both helper and gutless Adeno-associated virus 25 21 vectors have the same viral capsid, separation must be Other viruses 148 100 addressed before purification. Thus far, strategies have Others 183 148 been based on reducing the packaging efficiency of the helper genome compared to the gutless genome, either Data from www.wiley.co.uk/genmed/clinical. by mutating its packaging signal,18–20 by the different 0 10000 20000 30000 36000 MLP Ψ E1L1 L2 L3 L4 E3 L5 Ad5 genome ITR ITR E2B E2A E4 Ψ ∆E1 ∆E3 First generation Transgene Ψ ∆E1 ∆E2 ∆E3 ∆E4 Second generation Transgene Ψ Helper Dependent Ad Transgene Figure 1 Map of adenovirus serotype 5 genome and different generations of adenoviral vectors. Early transcripts are represented by E1–E4 regions and late transcripts are represented by L1–L5 regions. MLP: major late promoter; C: packaging signal. Gene Therapy Gutless adenovirus R Alba et al S20 size of its genome13 (genomes bigger or smaller than These levels of helper contamination seem to be due the optimal do not package efficiently), or by specific to the limited efficiency of packaging signal excision elimination of its packaging signal during viral produc- associated to low recombinase activity or with low tion.21 endogenous levels of recombinases. This is caused either Initial strategies consisted in the use of helper- by adenovirus-mediated host cell shut off or by dependent adenoviruses carrying a defective packaging cytotoxicity of high levels of Cre recombinase.22,23 signal.13 These vectors had extensive regions of deleted Although the final level of helper contamination is low, viral genome, but they still have some coding regions. further reduction is desirable to minimize any potential Cotransfection of a wild-type adenovirus as a helper toxicity associated with the helper virus, especially when with this gutless permitted to propagate both adeno- high doses are required.24 viruses.