- Email: [email protected]
Gene therapy—the gutless approach pays off
M E E T I N G R E P O RT S
Gene therapy – the gutless approach pays off MOLECULAR AND CELLULAR BIOLOGY OF GENE THERAPY, KEYSTONE SYMPOSIUM, KEYSTONE, USA, 19–25 JANUARY 1998. A consistent concern at past gene will begin to play an increasingly systems will vanish in the process of therapy meetings has been the so- significant role in other gene therapy making clinically useful vectors. called vector gap, the difference be- protocols. Moving to the clinical arena, one tween the ability to transfer and As Joe Glorioso (Univ. of Pitts- of the most interesting stories was preexpress genes in ideal (usually in burgh, USA) pointed out in his pres- sented by Jeffrey Isner (St Elizabeth’s vitro) conditions, and the experiences entation, the molecular biology and Medical Center, Boston, USA), who found in vivo, where gene transfer life cycle of HSV makes it attractive as recounted his experience of using and persistence of gene expression a gene delivery vector3. In particular, plasmid DNA, engineered to express are both usually severely limited1. its ability to persist in a latent state in VEGF, for the treatment of ischaemic One approach to overcoming these neurons while expressing an intra- limb disease5. While normal venous obstacles was discussed by Inder nuclear transcript makes it potentially grafting procedures can be sufficient Verma (Salk Inst., USA), who, having useful for the treatment of diseases of to keep ischaemic limbs alive, they are focused initially on retroviral vectors, the nervous system and cancer. For tu- generally not enough to avoid pain has now transferred his attentions to mour therapy using HSV there are two or tissue damage, and of course there the lentivirus family. The ability of established approaches: direct killing is a finite number of veins that can be lentivirus nucleic acid to cross the of HSV-infected cells with ganciclovir grafted. Working on the hypothesis nuclear membrane obviates the or transduction of tumour cells with a that expression of VEGF would prorequirement for membrane break- vector expressing cytokines. In vivo mote the growth of new blood vesdown during mitosis, and sels, up to 2 mg of plasmid hence allows infection of DNA was injected into four non-dividing cells. Verma’s sites in the limb. After sevgroup has constructed a eral days, ultrasound scans replication-defective vecdemonstrated growth of new tor derived from HIV, conblood vessels, and there was taining the VSV glycoproan obvious clinical improvetein. Initial experiments in ment. The reasons for the animals have demonstrated successful outcomes in these gene transfer into postclinical experiments are HSV is potentially a powerful tool for the treatment mitotic neurons, liver and probably the requirement for of diseases of the nervous system. Image kindly muscle cells, with exprestransient expression of the provided by Joe Glorioso, Univ. of Pittsburgh, USA. sion persisting up to six growth factor, and the fact that it is a secreted protein. months post-injection. Further in vivo experiments using Factor studies in Glorioso’s laboratory have The first FDA-approved clinical trial IX knockout (haemophiliac) mice demonstrated a synergistic effect be- in gene therapy was started more than demonstrated clear expression of tween these two approaches. Animal seven years ago, with the attempt to human Factor IX, which persisted models were set up using tumours introduce the ADA gene via a retrountil the appearance of a specific derived from human glioblastoma viral vector into patients suffering from antibody response, the epitaph to cells that are resistant to exogenous an enzymatic deficiency. The current many a gene transfer experiment. TNF. However, the tumours were sen- status of these trials was reported by Much interest was also sparked by sitive to HSV vectors expressing TNF, Donald Kohn (Children’s Hospital, the presentations on the second gener- and this effect was potentiated by the Los Angeles, USA) and Richard Morgan ation or helper-dependent adenovirus addition of ganciclovir. The bystander (NIH, Bethesda, USA). Examination of vectors, in which all viral genes are effect has been shown to play a crucial T-cells from these patients, who are all deleted, allowing a cloning capacity role in many therapeutic approaches, given supplementary PEG-ADA treatof up to 35 kb but, more importantly, although operating under a number of ment, has shown the presence of the thereby minimizing the probable im- guises4. One suggestion to augment ADA gene (albeit at a level of 1 in mune response against the vector this effect was the use of HSV vectors 10 000 cells) and multi-lineage gene itself2. Frank Graham (McMaster Univ., expressing connexin, a constituent of expression, leading to the attempt to Hamilton, Canada) described tech- gap junctions, to improve transport of withdraw drug treatment. However, niques for the generation of relatively ganciclovir between tumour cells. when this was tried, the patients sufpure populations of these helper-free Using this vector in vivo, a significant fered a severe depletion in T cells, virus particles, containing up to 1012 enhancement of tumour killing could and a concomitant increase in infecparticles ml⫺1. Variants of these vec- be demonstrated. tions. Following retreatment with the tors expressing leptin were used to Although other presentations ADA enzyme, the T-cell level intreat ob/ob mice and, using the robust demonstrated many elegant and creased and the infections cleared, assay of weight loss, Graham was able imaginative approaches to improving suggesting that immune functions in to demonstrate a clear biochemical gene delivery vectors, there is always these patients are dependent on the effect. Following these results, it is the nagging worry that seemingly exogenous PEG-ADA, not the translikely that ‘gutless’ adenovirus vectors attractive features of novel delivery ferred gene. It is clear that effective TIG APRIL 1998 VOL. 14 NO. 4 Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01439-5
136
M E E T I N G R E P O RT S therapy in this disease will require both increased transduction efficiency, and a higher level of expression of the transduced gene. Overall, this was a useful meeting, perhaps marking a turning point in the acceptance of gene therapy as an effective form of treatment. Certainly the mix of scientists from both academic and commercial backgrounds testified to the strength of interest in this area. The combination of excitement over the science, and the
rarefied mountain air, may have been responsible for the point, rather forcibly put by one eminent speaker, about the acceptability or otherwise of slides bearing corporate logos…
Further reading 1 Orkin, S.H. and Motulsky, A.G. (1995) Report and recommendations of the panel to assess the NIH investment in research on gene therapy, NIH press 2 Parks, R.J. et al. (1996) Proc. Natl.
Acad. Sci. U. S. A. 93, 13565–13570 3 Breakefield, X.O. et al. (1995) in The Internet Book of Gene Therapy, Appleton & Lange 4 Paillard, F. (1997) Hum. Gene Ther. 8, 1733–1735 5 Isner, J.M. et al. (1996) Lancet 348, 370–374
Nigel Whittle [email protected] Cantab Pharmaceuticals, 184 Science Park, Milton Road, Cambridge UK CB4 4GN.
MONITOR A ‘digest’ of some recent papers of interest in the primary journals
More is less Repeat-induced gene silencing in mammals Garrick, D., Fiering, S., Martin, D.I.K. and Whitelaw, E. Nat. Genet. 18, 56–59 Expression of transgenic constructs driven by promoter–enhancer combinations in mice can be a hit and miss affair. Expression levels depend on the position of transgene integration into the host genome and on transgene copy number. It has been difficult to assess the relative contributions of these two factors to the levels of transgene activity when making transgenic mice by conventional routes because neither variable can be evaluated independently. In this paper, however, Garrick et al. use Cre recombinase to derive low-copy sublines of transgenic mice from parental lines carrying >100 copies of a loxPbearing, erythroid-specific transgene.
Preserving the same location of the transgenes in low and high copy arrays thus enabled the effect of copy number on transgene expression to be addressed directly. Cre-induced, low copy transgenes were expressed 100–1000-fold more strongly than transgenes in the >100 copy parental lines. This suggests that large transgene arrays engage a copy-number-responsive silencing mechanism. Moreover, Garrick et al. showed that while the low copy transgenes are unmethylated and endonuclease sensitive, the high copy arrays are methylated and located in endonuclease-inaccessible chromatin. Nuclear run-on assays also confirmed that nascent transcripts
Numbing down Numb-associated kinase interacts with the phosphotyrosine binding domain of Numb and antagonizes the function of Numb in vivo Chien, N. et al. Mol. Cell. Biol. 18, 598–607 The asymmetrical localization of Numb is involved in cell fate decisions in neural and muscle precursors, where it antagonizes Notch activity. Chien et al. suggest that this is only part of the story. Using the yeast two-hybrid system they identified a serine–threonine kinase that interacts with Drosophila Numb, specifically with the highly conserved PTB (phosphotyrosine binding) domain. The
interaction is different from those of other proteins that bind to PTB domains because there is no requirement for a tyrosine phosphorylation site and, indeed, the Numb-associated kinase (NAK) only interacts with Drosophila and vertebrate Numb, not with other PTB domains tested. In the absence of mutants, the function of NAK is investigated in vivo using mis-expression. The TIG APRIL 1998 VOL. 14 NO. 4
137
from the low copy loci were more abundant than those from the high copy arrays. Position effects on transgene expression have been well-documented and this work now describes a repeatinduced silencing mechanism. It is unclear how the cell nucleus monitors its DNA for the presence of repeated elements, but this process might enable efficient silencing of mutagenic transposable elements and viruses. These results also reinforce the significance of locus control regions and insulator elements. These cis-acting elements can operate from within transgene arrays to overcome repeat-induced silencing and render expression of the arrays proportional to transgene copy number. Their dominant positive effects seem to determine the autonomy of transcriptionally active chromatin domains against a background of multiple heterochromatin-inducing mechanisms. ✍
phenotypes are the opposite of those seen with ectopic Numb expression, specifically in the sensory organ lineage there is a bias towards sheath cell fates in the embryo, and to hair or socket cell fates in the adult. These phenotypes are sensitive to the dosage of Numb, being enhanced in flies heterozygous for a Numb loss-of-function mutation and reduced in flies where Numb is also ectopically expressed The latter leads Chien et al. to hypothesize that NAK is involved in modifying the function of Numb once it is localized, rather than in the localization per se, and to speculate that NAK could be important in mediating the effects of Numb on Notch signalling. ✍
Gene therapy – the gutless approach pays off MOLECULAR AND CELLULAR BIOLOGY OF GENE THERAPY, KEYSTONE SYMPOSIUM, KEYSTONE, USA, 19–25 JANUARY 1998. A consistent concern at past gene will begin to play an increasingly systems will vanish in the process of therapy meetings has been the so- significant role in other gene therapy making clinically useful vectors. called vector gap, the difference be- protocols. Moving to the clinical arena, one tween the ability to transfer and As Joe Glorioso (Univ. of Pitts- of the most interesting stories was preexpress genes in ideal (usually in burgh, USA) pointed out in his pres- sented by Jeffrey Isner (St Elizabeth’s vitro) conditions, and the experiences entation, the molecular biology and Medical Center, Boston, USA), who found in vivo, where gene transfer life cycle of HSV makes it attractive as recounted his experience of using and persistence of gene expression a gene delivery vector3. In particular, plasmid DNA, engineered to express are both usually severely limited1. its ability to persist in a latent state in VEGF, for the treatment of ischaemic One approach to overcoming these neurons while expressing an intra- limb disease5. While normal venous obstacles was discussed by Inder nuclear transcript makes it potentially grafting procedures can be sufficient Verma (Salk Inst., USA), who, having useful for the treatment of diseases of to keep ischaemic limbs alive, they are focused initially on retroviral vectors, the nervous system and cancer. For tu- generally not enough to avoid pain has now transferred his attentions to mour therapy using HSV there are two or tissue damage, and of course there the lentivirus family. The ability of established approaches: direct killing is a finite number of veins that can be lentivirus nucleic acid to cross the of HSV-infected cells with ganciclovir grafted. Working on the hypothesis nuclear membrane obviates the or transduction of tumour cells with a that expression of VEGF would prorequirement for membrane break- vector expressing cytokines. In vivo mote the growth of new blood vesdown during mitosis, and sels, up to 2 mg of plasmid hence allows infection of DNA was injected into four non-dividing cells. Verma’s sites in the limb. After sevgroup has constructed a eral days, ultrasound scans replication-defective vecdemonstrated growth of new tor derived from HIV, conblood vessels, and there was taining the VSV glycoproan obvious clinical improvetein. Initial experiments in ment. The reasons for the animals have demonstrated successful outcomes in these gene transfer into postclinical experiments are HSV is potentially a powerful tool for the treatment mitotic neurons, liver and probably the requirement for of diseases of the nervous system. Image kindly muscle cells, with exprestransient expression of the provided by Joe Glorioso, Univ. of Pittsburgh, USA. sion persisting up to six growth factor, and the fact that it is a secreted protein. months post-injection. Further in vivo experiments using Factor studies in Glorioso’s laboratory have The first FDA-approved clinical trial IX knockout (haemophiliac) mice demonstrated a synergistic effect be- in gene therapy was started more than demonstrated clear expression of tween these two approaches. Animal seven years ago, with the attempt to human Factor IX, which persisted models were set up using tumours introduce the ADA gene via a retrountil the appearance of a specific derived from human glioblastoma viral vector into patients suffering from antibody response, the epitaph to cells that are resistant to exogenous an enzymatic deficiency. The current many a gene transfer experiment. TNF. However, the tumours were sen- status of these trials was reported by Much interest was also sparked by sitive to HSV vectors expressing TNF, Donald Kohn (Children’s Hospital, the presentations on the second gener- and this effect was potentiated by the Los Angeles, USA) and Richard Morgan ation or helper-dependent adenovirus addition of ganciclovir. The bystander (NIH, Bethesda, USA). Examination of vectors, in which all viral genes are effect has been shown to play a crucial T-cells from these patients, who are all deleted, allowing a cloning capacity role in many therapeutic approaches, given supplementary PEG-ADA treatof up to 35 kb but, more importantly, although operating under a number of ment, has shown the presence of the thereby minimizing the probable im- guises4. One suggestion to augment ADA gene (albeit at a level of 1 in mune response against the vector this effect was the use of HSV vectors 10 000 cells) and multi-lineage gene itself2. Frank Graham (McMaster Univ., expressing connexin, a constituent of expression, leading to the attempt to Hamilton, Canada) described tech- gap junctions, to improve transport of withdraw drug treatment. However, niques for the generation of relatively ganciclovir between tumour cells. when this was tried, the patients sufpure populations of these helper-free Using this vector in vivo, a significant fered a severe depletion in T cells, virus particles, containing up to 1012 enhancement of tumour killing could and a concomitant increase in infecparticles ml⫺1. Variants of these vec- be demonstrated. tions. Following retreatment with the tors expressing leptin were used to Although other presentations ADA enzyme, the T-cell level intreat ob/ob mice and, using the robust demonstrated many elegant and creased and the infections cleared, assay of weight loss, Graham was able imaginative approaches to improving suggesting that immune functions in to demonstrate a clear biochemical gene delivery vectors, there is always these patients are dependent on the effect. Following these results, it is the nagging worry that seemingly exogenous PEG-ADA, not the translikely that ‘gutless’ adenovirus vectors attractive features of novel delivery ferred gene. It is clear that effective TIG APRIL 1998 VOL. 14 NO. 4 Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01439-5
136
M E E T I N G R E P O RT S therapy in this disease will require both increased transduction efficiency, and a higher level of expression of the transduced gene. Overall, this was a useful meeting, perhaps marking a turning point in the acceptance of gene therapy as an effective form of treatment. Certainly the mix of scientists from both academic and commercial backgrounds testified to the strength of interest in this area. The combination of excitement over the science, and the
rarefied mountain air, may have been responsible for the point, rather forcibly put by one eminent speaker, about the acceptability or otherwise of slides bearing corporate logos…
Further reading 1 Orkin, S.H. and Motulsky, A.G. (1995) Report and recommendations of the panel to assess the NIH investment in research on gene therapy, NIH press 2 Parks, R.J. et al. (1996) Proc. Natl.
Acad. Sci. U. S. A. 93, 13565–13570 3 Breakefield, X.O. et al. (1995) in The Internet Book of Gene Therapy, Appleton & Lange 4 Paillard, F. (1997) Hum. Gene Ther. 8, 1733–1735 5 Isner, J.M. et al. (1996) Lancet 348, 370–374
Nigel Whittle [email protected] Cantab Pharmaceuticals, 184 Science Park, Milton Road, Cambridge UK CB4 4GN.
MONITOR A ‘digest’ of some recent papers of interest in the primary journals
More is less Repeat-induced gene silencing in mammals Garrick, D., Fiering, S., Martin, D.I.K. and Whitelaw, E. Nat. Genet. 18, 56–59 Expression of transgenic constructs driven by promoter–enhancer combinations in mice can be a hit and miss affair. Expression levels depend on the position of transgene integration into the host genome and on transgene copy number. It has been difficult to assess the relative contributions of these two factors to the levels of transgene activity when making transgenic mice by conventional routes because neither variable can be evaluated independently. In this paper, however, Garrick et al. use Cre recombinase to derive low-copy sublines of transgenic mice from parental lines carrying >100 copies of a loxPbearing, erythroid-specific transgene.
Preserving the same location of the transgenes in low and high copy arrays thus enabled the effect of copy number on transgene expression to be addressed directly. Cre-induced, low copy transgenes were expressed 100–1000-fold more strongly than transgenes in the >100 copy parental lines. This suggests that large transgene arrays engage a copy-number-responsive silencing mechanism. Moreover, Garrick et al. showed that while the low copy transgenes are unmethylated and endonuclease sensitive, the high copy arrays are methylated and located in endonuclease-inaccessible chromatin. Nuclear run-on assays also confirmed that nascent transcripts
Numbing down Numb-associated kinase interacts with the phosphotyrosine binding domain of Numb and antagonizes the function of Numb in vivo Chien, N. et al. Mol. Cell. Biol. 18, 598–607 The asymmetrical localization of Numb is involved in cell fate decisions in neural and muscle precursors, where it antagonizes Notch activity. Chien et al. suggest that this is only part of the story. Using the yeast two-hybrid system they identified a serine–threonine kinase that interacts with Drosophila Numb, specifically with the highly conserved PTB (phosphotyrosine binding) domain. The
interaction is different from those of other proteins that bind to PTB domains because there is no requirement for a tyrosine phosphorylation site and, indeed, the Numb-associated kinase (NAK) only interacts with Drosophila and vertebrate Numb, not with other PTB domains tested. In the absence of mutants, the function of NAK is investigated in vivo using mis-expression. The TIG APRIL 1998 VOL. 14 NO. 4
137
from the low copy loci were more abundant than those from the high copy arrays. Position effects on transgene expression have been well-documented and this work now describes a repeatinduced silencing mechanism. It is unclear how the cell nucleus monitors its DNA for the presence of repeated elements, but this process might enable efficient silencing of mutagenic transposable elements and viruses. These results also reinforce the significance of locus control regions and insulator elements. These cis-acting elements can operate from within transgene arrays to overcome repeat-induced silencing and render expression of the arrays proportional to transgene copy number. Their dominant positive effects seem to determine the autonomy of transcriptionally active chromatin domains against a background of multiple heterochromatin-inducing mechanisms. ✍
phenotypes are the opposite of those seen with ectopic Numb expression, specifically in the sensory organ lineage there is a bias towards sheath cell fates in the embryo, and to hair or socket cell fates in the adult. These phenotypes are sensitive to the dosage of Numb, being enhanced in flies heterozygous for a Numb loss-of-function mutation and reduced in flies where Numb is also ectopically expressed The latter leads Chien et al. to hypothesize that NAK is involved in modifying the function of Numb once it is localized, rather than in the localization per se, and to speculate that NAK could be important in mediating the effects of Numb on Notch signalling. ✍