Correcting a Deficiency (2001)

Most gene therapy research concerns gene addition strategies, where an exogenous gene, deprived of its normal chromosomal context and usually most of its intron/exon structure and regulatory elements, is required to produce sustained and appropriate expression of a therapeutic protein. Despite notable advances and successes, unstable or inappropriate gene expression remains a fundamental problem in gene addition strategies. In principle, however, these problems can be avoided by gene correction strategies in which an endogenous defective disease gene is repaired so as to regenerate a normal gene in its natural chromosomal context (1Yáñez R.J. Porter A.C.G. Therapeutic gene targeting.Gene Ther. 1998; 5: 149-159Crossref PubMed Scopus (142) Google Scholar). Gene correction has two further advantages over gene addition: it can overcome dominant genetic disease mutations and it avoids safety concerns associated with random integration of DNA into the genome. These are important attractions, so why do papers concerning gene correction, such as that by Inoue et al. in this issue (2Inoue N. Dong R. Hirata R.K. Russell D.W. Introduction of single base substitutions at homologous chromosomal sequences by adeno-associated virus vectors.Mol. Ther. 2001; 3: 526-530Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), appear so infrequently in the gene therapy journals? One reason is that gene correction is most directly applicable to inherited single gene disorders, and therefore of limited interest to the many researchers tackling complex or acquired genetic disorders, including cancer. But this alone cannot been the reason; the single gene disorders represent a huge amount of human suffering world-wide, and a routine effective gene therapy for just one of the most common disorders would be a major achievement. The main reason is probably one of efficiency: gene correction, e.g., by homologous recombination between a transfected, usually electroporated, DNA construct and the target disease gene (gene targeting), typically occurs at frequencies in the order of 1 in 105 transfected cells. This is in stark contrast to gene addition, which can be achieved with retroviral vectors, for example, at frequencies approaching 100% of treated cells. Add to this the fact that, in a clinical setting, the desired target cells are rare somatic stem cells that may make up only a tiny fraction of the treated cells, and gene correction begins to look like a lost cause. However, gene correction is too attractive an idea to be abandoned so easily and there are many labs whose work may lead to ways of improving its efficiency. These range from efforts to understand and manipulate the molecular machinery of DNA recombination and repair to studies of key variables such as host cell status, vector design and DNA delivery method. This last variable might seem an ideal candidate for optimization because the inefficiency of electroporation is clearly a contributory factor to low targeting frequencies. But alternative delivery methods, both viral (retroviral, adenoviral) and nonviral (calcium phosphate precipitation, lipofection and nuclear microinjection), have so far failed to offer any major practical improvements. The discovery that adeno-associated virus (AAV) can be used for gene targeting in normal human cells with efficiencies of up to 1% of infected cells was therefore a significant development (3Russell D.W. Hirata R.K. Human gene targeting by viral vectors.Nat. Genet. 1998; 18: 325-330Crossref PubMed Scopus (267) Google Scholar). Control experiments demonstrated that this represented an improvement of 2 to 3 logs over targeting by transfection of naked DNA. Russell and colleagues went on to show AAV to be capable of introducing any of a range of genetic alterations into a variety of different target loci and with high fidelity. Targeting efficiency correlated directly with both the multiplicity of infection and the amount of homology between vector and target, but there was still considerable variation in targeting efficiencies when these variables were held constant. In the latest paper from this group, Inoue et al. explore the possibility that at least some of this variation may be related to the type of base-pair substitution being introduced (2Inoue N. Dong R. Hirata R.K. Russell D.W. Introduction of single base substitutions at homologous chromosomal sequences by adeno-associated virus vectors.Mol. Ther. 2001; 3: 526-530Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Six AAV vectors were made, each introducing a different base-pair substitution into the splice acceptor site of exon 2 of the human HPRT gene and a sixfold range in frequency of gene targeting was observed. These results can be interpreted, at least partly, in terms of a known bias in the mismatch repair machinery, which recognizes base-pair mismatches that occur in recombination intermediates. However, similar experiments by the same group involving a different target locus showed little variation in targeting efficiency with choice of base-pair substitution. The reasons for this discrepancy are unclear; presumably other factors such as sequence and chromatin context also influence gene correction efficiency. Whatever such factors maybe, the observation that the complete set of substitutions could be successfully introduced by AAV into a target gene is an important one if AAV is ever to be developed as therapeutic gene correction vector. Encouraging as these studies are, without progress in the isolation and growth of somatic stem cells, or in the exciting possibilities for combining gene correction with nuclear transfer and in vitro stem cell differentiation (4Gurdon J.B. Colman A. The future of cloning.Nature. 1999; 402: 743-746Crossref PubMed Scopus (98) Google Scholar), further improvements in targeting efficiency are desirable before AAV-mediated somatic gene correction strategies can move to the clinic. Preliminary experiments in a model system, such as delivery to mouse hematopoietic precursor cells, for which targeted gene modification by an electroporated vector was recently demonstrated (5Hatada S. Nikkuni K. Bentley S.A. Kirby S. Smithies O. Gene correction in hematopoietic progenitor cells by homologous recombination.Proc. Natl. Acad. Sci. USA. 2000; 97: 13807-13811Crossref PubMed Scopus (40) Google Scholar), would seem an appropriate next step. Experiments to understand what makes AAV a good gene correction vector would also be valuable. Is it simply that very high copy numbers can be delivered to the host cell nucleus or something about the DNA genome of AAV, such as its single-strandedness or hairpin ends? Answers to these kinds of questions may lead to improvements in the design not only of future AAV vectors but also of gene correction vectors in general. Finally, in the area of targeted mutagenesis, which is relevant to gene therapy as a way to establish animal models of human disease and elucidate disease gene function, AAV-mediated gene targeting has yet to catch on. However, this could change if the skill and experience required for making the necessary high-titer AAV stocks become more widely available. Certainly, there is plenty to be done in gene correction research, AAV-mediated or otherwise, but also good reasons to be optimistic. Perhaps the scarcity of gene correction papers in gene therapy journals is another deficiency we can look forward to correcting.