12 |
DISCUSSION |
Nir I, Papermaster D 1986 Opsin gene expression during early and late phases of retinal degeneration in rds mice. Exp Eye Res 51:257^267
Ovchinnikov YA, Abdulaev NG, Feigina MY et al 1982 The complete amino acid sequence of visual rhodopsin. Bioorg Khim 8:1011^1014
Peterson WM, Wang Q, Tzekova R, Wiegand SJ 2000 Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia. J Neurosci 20:4081^4090 Redmond TM, Yu S, Lee E et al 1998 Rpe65 is necessary for production of 11-cis Vitamin A in
the retinal visual cycle. Nat Genet 20:344^351
Sohocki MM, Daiger SP, Bowne SJ et al 2001 Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Hum Mutat 17:42^51
Stahl N, Yancopoulos GD 1994 The tripartite CNTF receptor complex: activation and signaling involves components shared with other cytokines. J Neurobiol 25:1454^1466
Travis GH, Brennan MB, Danielson PE, Kozak CA, Sutcli¡e JG 1989 Identi¢cation of a photoreceptor-speci¢c mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature 338:70^73
Travis GH, Sutcli¡e JG, Bok D 1991 The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein. Neuron 6:61^70
Usukura J, Bok D 1987 Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay. Exp Eye Res 45:501^515
Vollrath D, Feng W, Duncan JL et al 2001 Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proc Natl Acad Sci USA 98:12584^12589
Wahlin KJ, Campochiaro PA, Zack DJ, Adler R 2000 Neurotrophic factors cause activation of intracellular signaling pathways in Mˇller cells and other cells of the inner retina, but not photoreceptors. Invest Ophthalmol Vis Sci 41:927^936
DISCUSSION
[Note added in press by William Hauswirth: Recently, the issue of vector AAV chromosomal integration has arisen as a potential source of tumorigenesis in light of recent reports of retrovirus vector integration-related leukaemias in three of 10 treated X-linked SCIDS patients. First, it is important to emphasize that the biology of integration is very di¡erent between AAV and retroviral vectors, and that it is a mistake to draw an analogy between the two. Retroviruses lead to passenger gene expression through an obligate integration event; hence, 100% chromosomal integration is found. Additionally, retroviruses integrate relatively site speci¢cally near a variety of proor anti-tumour genes, as they did in the XSCIDS cases. In contrast, AAV vectors from which e¡ective passenger gene expression emanates are very rarely integrated (less than 0.5% of vector genomes, Schnepp et al 2003). This recent study con¢rms many previous, but less quantitative studies, also ¢nding very low frequencies of integration and large amounts non-integrated vector from which expression originated (labs of Flotte, Englehardt, Xiao, Samulski). Even when the experiment is purposely biased to favour detection of integrated AAV vector, integration events were still rare (Check 2003). Finally, in over 100 patients treated to date with AAV vectors over the past 5+ years, no vector related tumours have been reported. Clearly, caution
GENE THERAPY |
13 |
dictates that the tumour incidence in preclinical and clinical AAV gene therapy trials continues to be carefully monitored, however it is a mistake, and perhaps a disservice to the patient population, to make simple extrapolations from one vector type to another without a clear understanding of the biological di¡erences between them.]
McInnes: The retrovirus promoters are very powerful and have distal e¡ects. Do the promoters present in AAV have the same potential risk?
Ali: They are much weaker.
Hauswirth: This is probably because of the inverted terminal repeats that can act as silencers and/or insulators for transcription either coming into the region of going out of the region.
Ali: It may be fair to speculate that retroviral transduction of a proliferating population of cells is more likely to lead to oncogenic events than transduction of non-dividing tissue. It has been suggested that the parts of the genome that are available for integration are composed of DNaseI-sensitive sites, which implies that these are genes that are being expressed. If we think about the stem cells that were being targeted, the sorts of genes that are being expressed are possibly genes involved in cell division and cell regulation. This may not be the case in neuronal cells and photoreceptors. I agree with Dean Bok that issues surrounding integration are important . we need to understand this and be rigorous about it . but I think there may be good reasons for thinking that integration events in the retina may not present the same problem as those in proliferating stem cells.
Hauswirth: As Dean implied, if you add back the viral rep gene, which is the gene that mediates site-speci¢c integration in chromosome 19 in the wild-type, this halves the amount of space available for your own gene. This leaves just 2.2 kb, which is not enough for many of the things we want to do. It has its own problems. One idea that gets us up to 5 kb of payload in the vector is to put in two viruses, one expressing the rep gene and one expressing the therapeutic gene. Presumably rep will act to target the therapeutic gene to chromosome 19, which we know is a safe site since 60% of the world’s population has AAV integrated into this site.
LaVail: Can you get the titres high enough so that you can be sure that you have a doubly infected cell?
Hauswirth: This is probably not a problem. A back of the envelope calculation would suggest we have done this in several inducible situations. With a Doxinducible system the multiplicity of infections is typically over 1000 per photoreceptor cell, so the odds of getting two vectors into one cell are very high.
McInnes: Might the choice of CNTF as the ¢rst neurotrophic factor to use in therapy retrospectively turn out to be unfortunate? Perhaps the e¡ects it is having on the cell might not occur with other factors.
14 |
DISCUSSION |
LaVail: That is a possibility. On the other hand, it is very hard to turn your back on a molecule that shows a positive rescue e¡ect in over 13 di¡erent forms of inherited retinal degeneration. It needs to be given every chance to work. We need to see what these negative e¡ects have on the structure of the cell, and the rami¢cations of this for vision over the long term. Some people have suggested it may be that the lowered amplitude of the output of the cell is actually an attempt by the photoreceptor cell to preserve its energy level and live longer under unusual circumstances. One of the points you make is that even though there is a ¢nite number of retinal vision researchers, we do need to look at some of the other neurotrophic factors. There is, however, clearly a speci¢city here. At ¢rst, when people began using CNTF with better delivery systems as opposed to bolus injections, it appeared that CNTF provided rescue in every case. Thus, it appeared that all our negative ¢ndings were artifacts of the bolus injection. Does every agent work in every degeneration? I think the answer is no, on the basis of Don Zack’s and Peter Campochiaro’s work in transgenic animals (Yamada et al 2001) and some of our studies (M. M. LaVail, unpublished results). Certain agents need to be looked at more closely than they have been.
McInnes: It is clear that these cells might be cycling. Has anyone done labelling? LaVail: We have never seen mitotic ¢gures, but the photoreceptor cells are clearly more immature. There are studies showing that CNTF can impair
di¡erentiation and inhibit opsin expression.
Bok: We have hammered these cells as hard as we can. When Bill Hauswirth engineered this CNTF vector that we employed in our study, he even introduced a couple of amino acid changes that increased the a⁄nity of the CNTF for the a subunit of its receptor. We are now cutting back on the dose to see whether we can maintain the rescue e¡ect but get rid of the so-called ‘side e¡ects’. We are turning on a lot of di¡erent genes in these cells and this somehow impacts on the phototransduction cascade in ways that we don’t understand.
Zack: Rod McInnes mentioned that one approach is to look at other factors. But there’s another approach with CNTF: how about looking at cells other than photoreceptors? CNTF acts on a number of other neuronal cells. Have people studying these other neuronal cells seen evidence for change in gene expression with CNTF?
Bok: Looking at the retinas we did not perceive changes in other retinal neurons. But we really haven’t explored this.
Zack: In spinal cord injury, is there evidence that CNTF is changing the neuronal cells or is it just leading to their better survival?
LaVail: In general, I don’t think the other ¢elds are nearly as far along as we are. For them it isn’t as easy to look at the tissues, and they don’t know exactly where they are injecting.
GENE THERAPY |
15 |
Farber: Adriana Di Polo has done beautiful work with CNTF on ganglion cells and she gets great rescue.
Zack: But has she looked at the function of the ganglion cells? The rescue is the ¢rst part, but it is the function that we need to maintain.
Aguirre: With regard to CNTF and the changes in the photoreceptor cells, we found marked reactive changes in the dog retina following FGF2 and FGF18 administered with AAV vectors. Although there was dramatic rescue e¡ect in terms of the number of rows of outer nuclear layer (ONL) nuclei remaining, in some areas there were more nuclei when we ended than the number present at the time of injection. This suggested that there was an increased proliferation. Unlike the CNTF experiments in the rcd1 retinas where rescue was dramatic and the structural preservation was quite good, the rescue was accompanied by marked abnormalities. Personally, I am not that concerned that there is a decrease in the b-wave following CNTF administration. Some of the patients with Duchenne’s muscular dystrophy have a b-wave response that is changed towards a negative type ERG, but they don’t report any changes in night vision defects. If you have a very positive rescue e¡ect and there is a slight decrease in b-wave amplitude, this might not be clinically signi¢cant.
Swaroop: What do we know about CNTF’s downstream pathways? Would it be an option to use some of these downstream molecules as targets for future drug discovery? What is known about CNTF function in the retina? Can we modulate downstream signalling molecule(s) that would still have the bene¢cial e¡ects of CNTF with fewer side e¡ects?
LaVail: Some people have been looking at that, but it is a little too early to make any conclusions. One of the problems is that there is a dose-dependent side e¡ect of CNTF just as there is a dose-dependent rescue e¡ect. We need to know about this for every single aspect of the rescue process.
Farber: Dean Bok, you talk about all these retinal degenerations in mice, rats and dogs. What has been done in this respect with primates?
Bok: There are very few studies of this type in primates, because we don’t have the models. But we can now more readily create these models using knock-down experiments with vectors or by introducing mutations. There is a colony of monkeys that Paul Sieving might be interested in trying to bring to the USA from Japan. They have what seems to be a macular degeneration.
Zack: There is an interesting reverse parallel. In the glaucoma ¢eld they have a very good monkey model but limited rodent models. We are blessed with good rodent models but we are struggling for a monkey one.
References
Check E 2003 Harmful potential of viral vectors fuels doubts over gene therapy. Nature 423:573^574
16 |
DISCUSSION |
Schnepp BC, Clark KR, Klemanski DL, Pacak CA, Johnson PR 2003 |
Genetic fate |
of recombinant adeno-associated virus vector genomes in muscle. J Virol 77: 3495^3504
Yamada H, Yamada E, Ando A et al 2001 Fibroblast growth factor-2 decreases hyperoxiainduced photoreceptor cell death in mice. Am J Pathol 159:1113^1120