Gene therapy for haemophilia (2001)

The possibility of a ‘cure’ for haemophilia A and B remains the ultimate goal for patients with haemophilia. The cloning of the factor VIII and IX genes in the 1980s and subsequent production and introduction of recombinant factor VIII and IX over the last decade have continued to promise the prospect of a gene therapy treatment for haemophilia. In recent years, much work has been focused on developing strategies for gene therapy treatments, and we are now seeing phase I trials for both haemophilia A and B. How close are we to realizing our goal? Gene therapy, in the broadest sense, is the introduction of foreign genetic material into a cell with therapeutic intent. The ultimate gene therapy for haemophilia A and B would be therapeutic gene replacement and/or direct correction of the molecular defect in the mutated factor VIII or IX gene. Direct genomic modification, using chimeric RNA/DNA oligonucleotides (Kren et al, 1998, 1999), has been demonstrated, but for haemophilia A and B therapeutic gene replacement and/or direct correction of the molecular defect remain a long way in the future for clinical application. Gene therapy for haemophilia today therefore relies upon the addition of normal functional coagulation factor DNA sequences with appropriate promoter and enhancer elements to the cells of a patient with a defective coagulation factor gene, so that the modified cells can produce functional protein as directed by the inserted DNA. Although the haemophilias have long been seen as a good clinical model for the development of gene therapy for single-gene disorders, it is now a major target for gene therapy, as it has several fundamental advantages over other single-gene disorders: there is a simple cause and effect relationship between coagulation factor deficiency and disease phenotype/clinical symptoms. Tissue-specific expression of the transgene and precise regulation are probably unimportant; suitable well-characterized small and large animal models are available for preclinical studies. In clinical trials, efficacy can be established and assessed easily using both clinical and laboratory end-points. From a clinical point of view, haemophilia gene therapy would be a major step forward. Replacement therapy with factor VIII and IX products is now safer than ever but continues to be less than ideal. Although there is increasing use of recombinant products, with virus-inactivated plasma products, there remain ever-present concerns about blood-borne infection and contamination with infectious prions causing variant Creutzfeldt–Jakob disease (vCJD) (Will et al, 1996; Evatt, 1998). Even with state-of-the-art prophylactic regimes, replacement treatment must be administered intravenously at frequent intervals, which is often problematic in young children. There have been major advances in the field in recent years but, with present technology, gene therapy is unlikely to be able to provide a total ‘cure’ for haemophilia. Technology presently available is still not sufficiently advanced to effect a lifelong gene transfer that ensures continuing production of coagulation factor to normalize levels. Despite this, perhaps the most important single aspect of haemophilia that makes it such an excellent target for gene therapy is the fact that even a small rise (1–2% of physiological levels) in circulating factor VIII or IX would have a significant beneficial therapeutic effect, protecting against spontaneous bleeding and potentially transforming the lives of patients with severe haemophilia. In essence, endogenous factor production to afford such small rises in factor levels would achieve the goals of prophylaxis without regular infusions of concentrate. Although in concept gene therapy is equally applicable to haemophilia A and B, there are some important differences in terms of the practical aspects of developing a gene therapy treatment for each disorder. Factor IX cDNA is only 3 kb, whereas factor VIII cDNA is significantly larger at 8·8 kb. Smaller partially deleted factor VIII genes that do not contain the large internal B domain of the protein reduce the size of the factor VIII cDNA by ≈ 30%. Such B-domain-deleted factor VIII remains fully functional in vivo, as the B domain is not required for coagulant activity (Berntorp, 1997). However, the large size of the factor VIII gene has placed constraints upon the choice of gene delivery system for use in haemophilia A models and has led to different approaches being developed for haemophilia A and B. In addition, although a 1–2% increase in plasma level would have beneficial effects in both diseases, it must be borne in mind that the normal plasma level of factor IX (5 µg/ml) is some 50 times higher than for factor VIII (100 ng/ml). Successful gene therapy approaches for haemophilia B may therefore not work well for haemophilia A and vice versa. 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The cDNA at 8·8 is large to be by a B-domain-deleted has been in the of at gene therapy for haemophilia of the B domain has the of increasing factor VIII with the only is factor VIII cDNA a by of but it is to contain the that in levels of factor VIII et al, et al, et al, et al, sequences et al, et al, whereas et al, et al, effect is to be by sequences the factor VIII cDNA that are a with seen in sequences in Such have been in the of and et al, et al, Factor VIII is a large and protein that is to the it is to factor it is to factor VIII gene therapy to be factor VIII must be into the to to circulating of target and cells to factor VIII is than for factor IX and haemophilia B. with factor expression of factor VIII has been in a of cell and cell has been by that functional factor VIII be in cell transfer of factor VIII into et al, cells et al, and et al, However, levels of production to than and expression of factor VIII frequent to be the of the sequences in the factor VIII as of such sequences to the production of or the expression of factor However, of an of the factor VIII to significant in both and factor VIII expression et al, et al, of for haemophilia A gene therapy an with of and et al, to produce factor However, using an into factor VIII close to normal in et al, In the as of with modified into and on the of of that the and of the such as target cell and important in factor VIII levels in the factor VIII expression for gene therapy has been seen as an is to and be by of with factor has been to produce levels of factor VIII in et al, produce factor VIII activity of to Factor VIII expression as a of promoter et al, may be an for haemophilia A gene therapy, although choice of promoter to be In gene therapy, to target is the major target for work has focused on to possibility and to be and to in vivo, the cells to be or to However, other important that have to are a effect of and of a phase between and factor VIII production in and there a possibility of an in gene of are now that are than In addition, in the of the has for The use of that or the of has been significantly to and may have an increasing in developing gene therapy et al, 1996; et al, 1998). factor VIII have been into haemophilia A et al, Factor VIII levels of physiological in ≈ of the with some levels of factor The developed In factor VIII expression for than and for the that haemophilia A be in an animal model by a gene addition The expression of factor VIII in in the and may from higher in young with in has been in with expression of factor ≈ of normal for levels in the with the probably of higher with haemophilia have been with system using a factor VIII gene of the VIII in model system has although production of factor not to of that may be in haemophilia have been for factor VIII gene therapy of B-domain-deleted factor VIII in and in of Although other may be the use of promoter elements has the of expression to the factor VIII production is seen in normal et al, with normal factor VIII levels in some correction has been in et al, and et al, In expression has been for over using et al, and et al, factor VIII However, in expression is et al, of a to factor factor VIII has been in the haemophilia A model using an but led to expression as a of transgene and et al, in have that a factor VIII is at therapeutic levels of an and et al, The of expression in model is not as the only and it that factor VIII only at higher of of of and and at the has been for factor et al, factor VIII expression using to have a effect in vivo, of effects or et al, would have important for the therapeutic of for clinical The of a and the expression of the factor VIII transgene in the system to the use of the that sequences have been only that are required and the factor VIII cDNA the of a promoter et al, a at or led to expression of factor VIII in at physiological levels with in some as a of the production of factor VIII although it in that significant A effect to in have the expression of levels of factor VIII to and correction of the in a et al, However, and of for haemophilia A been seen as problematic of the small of and the large size of the factor VIII gene. an the factor VIII gene by using of and et al, The in of to produce than physiological levels of factor VIII ng/ml). Although remain as to production of or in cells not has effect on and the development of to factor A would be the of a factor VIII expression that and not production or transgene has been a major B-domain-deleted factor VIII have been developed that can be in although are larger than and et al, et al, of such factor VIII in severe led to the production of factor VIII with plasma levels ≈ 50 and factor VIII in et al, long provide for the use of for haemophilia A as well as haemophilia B. 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Direct genomic modification, using chimeric RNA/DNA oligonucleotides to the factor IX gene, has been in (Kren et al, as has correction of model gene in (Kren et al, there is a large a of in such approaches would of therapy for each patient and would not the in severe haemophilia However, has in that it may be to mutated genes to haemophilia. genetic material is the therapeutic gene therapy may be seen as different from other of However, gene therapy a in the of to Gene therapy has been seen to be applicable to for which therapeutic approaches are or the for treatment to be Although and the and that today haemophilia may the and that in clinical the patients most to from a gene therapy to treatment are with severe haemophilia level However, other a be seen to have the most to from a gene therapy However, is it to a and potentially treatment on young is important to the and of to such a potentially treatment with and for in clinical trials be gene therapy is to be and and with gene for each to the of target and that in the each the of of the of and to the patient to future must be and against and may therefore between clinical patients be with factor have or of and have a molecular defect for of The of such as and in is with infection are may not and a would the of patients with infection with may be suitable patients on therapy in to in or gene therapy to infection in gene The of in and upon the of disease is trials be to are or other there is on in to the is not an and other for may phase clinical trials for haemophilia A and B have been and use a of gene transfer and Although have been to has been so significantly as to to in the The to be have been using an of factor of factor IX in patients with severe haemophilia B and is on the preclinical animal work In the patients so at increasing in has for of of sequences production of IX et al, et al, the of and and the expression of factor IX in and the in plasma factor IX level in of the to but factor IX and a in the of factor IX infusions in of the et et al, from have that on animal may have the required in to achieve therapeutic levels. A is using an haemophilia are presently being using of and in In the cells are from the patient, and with a B-domain-deleted factor VIII gene by A of cells that factor VIII is isolated of the cells into the have and expression of factor VIII of for in a single treatment et al, although preclinical are to be patients have been with cells administered by with a of et al, There have been or VIII of the patients factor VIII activity levels increase in activity on In the of patients of and increase in factor VIII activity with a bleeding and factor VIII use et al, in factor VIII level Although being an modification, as a therapy, system is and would be to to large patient The haemophilia A clinical way an B-domain-deleted factor In is intravenously for is on the preclinical efficacy from the use of in and in patients at the in the significant and of Although levels patients factor VIII on at levels in the of although isolated higher levels for et al, A phase I using has patient to is on of the and over patients with haemophilia have been with gene therapy have to that therapeutic levels of factor VIII and IX can be However, to have not significant is most that present even it is most that such at to only in circulating factor VIII and IX and patients to treatment with for and is of that have been by animal trials that have higher rises in plasma factor VIII and IX than have been seen in the that animal can only be a to preclinical have that therapeutic levels of factor VIII or factor IX can be in both and advances in and gene transfer promise for the development of gene therapy for haemophilia A and B. Although gene therapy strategies on or expression of factor VIII or factor have now such that can models of haemophilia achieve therapeutic factor VIII levels in and correction of haemophilia has been a of the to and expression we a phase I clinical in severe haemophilia A patients using Although seen as a upon the continued of and of be can be for the treatment of haemophilia. remains the most in in However, the continuing development of and is Despite the use of expression that of and reduce the against gene products continues to which do not contain may provide a to although are to be fully as in the model et al, The use of for haemophilia gene therapy has been for factor IX and now for factor A of haemophilia B can be by of factor IX and correction of haemophilia B by or of factor IX The phase I clinical of of factor IX has in clinical which is phase I using an are In clinical a continuing remains the in patients with gene therapy The of development has been in animal models by the use of genes that has led to the production of the increasing use of genes in animal models of may now be partially However, in the gene therapy would increase or the of with therapy is remain that or in may it is not or not gene therapy in be of factor VIII or IX is from and potentially in with I and major at However, than to or an that production of factor VIII and factor IX in gene therapy may The of gene therapy for haemophilia in the been of gene therapy has led to the but that gene therapy in broadest in gene therapy for haemophilia is now with the of it can However, the in the field over the years, such as the remain and clinical trials now of with modified is haemophilia gene therapy trials are patient must remain a In correction of haemophilia A and B models is et al, et al, and the prospect of of haemophilia However, advances in gene it to into the in is to that such may promise the possibility of the coagulation factor gene in the of patients and so haemophilia in future Although perhaps an goal at the of aspects of gene therapy to be fully and as have for genetic in et al, of the is without to therapy for haemophilia. haemophilia remains a and in Even with the increasing of factor approaches using replacement therapy are in of the not of and Gene therapy, available as a administered a target therefore be gene therapy that are applicable to the not with haemophilia at a that can be