| Human Molecular Genetics | Pages |
Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration
Introduction
Results And Discussion
Materials And Methods
Construction of pAd5[beta]PDE, pAd5[beta][Mgr] and purification of EAMs
EAM and helper virus titers
Genomic structure of Ad5[beta]PDE
Administration of EAMs
Analyses of [beta]-PDE expression
Histology and [beta]-galactosidase expression
Detection of wild-type virus
Acknowledgements
References
Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration
INTRODUCTION
Adenovirus-mediated gene transfer is a promising technology for the treatment of human genetic diseases. Whereas adenovirus vectors have several advantages over other vector systems, limitations have become apparent with their use in vivo. These limitations include short-term expression of the delivered gene and loss of transduced cells. Despite the deletion of genes E1a and E1b in the so-called `first generation' adenoviral vectors, production of immunogenic viral proteins in transduced cells leads to MHC class I and class II mediated display of viral peptides on the surface of infected cells, targeting these cells for T-cell mediated clearance. Re-administration of adenoviruses to restore transgene expression is inefficient because of the production of neutralizing antiviral antibodies (1,2). In order to overcome some of these problems, `second generation' viral vectors containing deletions in other regions of the Ad genome (such as E2a and E4) have been developed; however, there is some uncertainty whether these vectors result in improved transgene persistence in animals (3-6). We (7,8) and others (9-13) have recently described a novel class of adenoviral vectors from which almost the entire Ad genome has been deleted [encapsidated adenovirus mini-chromosomes (EAMs)]. Our initial studies indicated that EAMs can be used to transduce cells in vitro (7). As our first step towards the use of EAMs in vivo, we describe EAM-mediated transduction of cells from the neurosensory retina. In particular, we show that this novel vector system can be used to transduce and rescue degenerating photoreceptor cells in the retinal degeneration (rd) mouse, a model for retinitis pigmentosa (RP) in humans.
RP is the name given to an array of disorders characterized by night blindness, progressive constriction of the visual field, an abnormal or non-detectable electroretinogram and photoreceptor degeneration. The disease may be inherited in an autosomal-dominant, autosomal-recessive, X-linked or digenic manner. Prevalence estimates range from 1 in 3000 to 1 in 6000, making RP one of the most common inherited forms of retinal disorders in humans. Some forms of RP in humans (14,15), mice (16,17) and dogs (18,19) have been associated with mutations in the gene encoding the [beta] subunit of guanosine 3[prime],5[prime]-monophosphate (cyclic GMP) phosphodiesterase (PDE).
A nonsense mutation (17) and/or an Xmv-28 proviral insertion (20) in the gene encoding [beta]-PDE causes retinal degeneration in the rd mouse, resulting in an aberrantly spliced mRNA and/or a predicted truncated protein product (21). Similarly, a nonsense mutation in the [beta]-PDE gene has been found in the Irish Setter dog (19). Homozygous rd mice undergo complete degeneration of photoreceptors within the first three weeks postnatal. We have shown previously that expression of [beta]-PDE in transgenic rd mice rescues photoreceptor degeneration (22). Moreover, heterozygous rd mice enjoy unimpaired vision, indicating that the mutation results in a lack of function, making the rd mouse and its equivalent in humans an excellent candidate for somatic gene therapy.
Several groups have demonstrated previously the efficacy of adenovirus as a gene transfer vector for the retina (23-26). In each of these investigations it has been demonstrated that a first-generation adenoviral vector is effective at delivering the Escherichia coli Lac Z gene regulated by a CMV promoter to the retina. In addition, first generation adenovirus vectors have been used to deliver ciliary neurotrophic factor or a [beta]-PDE cDNA regulated by a CMV promoter to the rd retina (27,28). The length of expression and rescue of degenerating rd retina using these vectors was transient, lasting up to 6 weeks (28). Nonetheless, these studies demonstrated an important `proof of principle' for gene therapy of RP. These studies taken together with data from other organ systems suggest that we need to develop and test improved vector systems. In order to further our potential for developing a gene-based therapy for RP, we have constructed an EAM containing the [beta]-PDE cDNA and delivered it to the subretinal space of homozygous rd mice. We have determined the expression of the transgene by RT-PCR, western blot analysis and PDE activity assays. Phenotypic rescue of the disease phenotype was determined by histological analyses. Our results indicate that EAMs may provide a significantly improved avenue for treatment of RP when compared with first-generation adenoviral vectors and may eventually contribute towards a gene therapy for RP.
RESULTS AND DISCUSSION
In contrast to most tissues, Ad-mediated gene therapy for vertebrate photoreceptors is particularly favorable from an immunological perspective. Certain organs such as testis, ovary, pregnant uterus and the eye are immunologically tolerant to foreign antigens, and are considered sites of immune privilege (29). Inflammatory cells entering the anterior chamber of the eye in response to viral infection undergo Fas-ligand induced apoptosis (30). However, a small number of inflammatory cells are associated with virally infected retina or subretinal space, indicating that these particular sites are only immunologically sequestered (30,31). As a consequence, preliminary gene therapy applications using first-generation adenovirus vectors to rescue the rd phenotype have resulted in minimal or physiologically insignificant levels of PDE activity. Recently, it has been demonstrated that a first-generation adenoviral vector encoding the [beta] subunit of PDE driven by the CMV promoter can rescue photoreceptors in the rd mouse for a period of ~6 weeks (28). We have investigated the use of a vector not able to express any Ad-encoded proteins to determine if the length of transgene expression can be extended with respect to first generation viral vectors. These studies will aid in our efforts to eventually determine an efficacious treatment of retinal diseases.
We have shown previously that a circular molecule containing fused embedded adenovirus inverted terminal repeats (origins of replication) and five packaging elements is replicated and packaged into mature virions in the presence of helper virus. The activity of an embedded origin of replication leads to the generation of linear mini-chromosomes from circular molecules (7). Ad DNA is packaged into virions in vivo in a polar, left-to-right fashion and is dependent on cis-acting packaging elements. The Ad5 packaging domain extends from nucleotide 194 to 358 and is composed of at least five distinct elements that are functionally redundant (32,33). These mini-chromosomes (34) or EAMs have a theoretical cloning capacity of 36 kb. We have shown previously that EAMs containing up to 22 kb of non-viral exogenous DNA can be used to transfer multiple gene-expression cassettes to cells in culture (7). In this study we have constructed an EAM consisting of a full-length (2.75 kb) murine [beta]-PDE cDNA regulated by a human [beta]-PDE promoter (350 bp), an E.coli Lac Z gene regulated by a cytomegalovirus promoter and the Ad5 origin of replication together with five cis-acting viral packaging signals-pAd5[beta]PDE (Fig.
Figure 1. Structure of pAD5[beta]PDE. The [beta] subunit of Mus musculus cGMP-phosphodiesterase (m[beta]PDE) and the E.coli [beta]-galactosidase (LacZ) cDNAs are regulated by a human [beta]-PDE (hPDE) and cytomegalovirus (CMV) enhancer/promoter, respectively. The [beta]-PDE cDNA and LacZ gene are polyadenylated by an SV40 early (E) and late (L) signal, respectively. The bacterial origin of DNA replication (ori E coli) and ampicillin selection (AmpR) for propagation in bacteria are encoded by pBSIIKS (Stratagene). The fused adenovirus origins of replication are represented by a left and right inverted terminal repeat (LITR/RITR). Following replication, encapsidation of DNA into mature capsids is accomplished by the five packaging signals ([Psi][Igr]-[Psi]V). Cotransfection of pAd5[beta]PDE with linear adenoviral DNA into human embryonic kidney 293 packaging cells produces EAMs and helper virions ~6-12 days post-transfection, as evidenced by the appearance of a cytopathic effect. The EAMs are propagated and amplified by six serial passages in 293 cells with a trans-acting E1/E3-deleted helper virus expressing human placental alkaline phosphatase (hpAP). The choice of hpAP as a helper allows monitoring of the relative populations of helper virus and EAMs at the various stages of serial propagation (Fig. Figure 2. Amplification of Ad5[beta]PDE. Following cotransfection (T) of 293 cells (vertical bars) with 5 µg of pAD5[beta]PDE and 0.5 µg of linear hpAP DNA, six serial passages were performed to amplify both AD5[beta]PDE and hpAP virions. Assays detecting [beta]-galactosidase (BG) or alkaline phosphatase (AP) activities were used to determine the total input (I) or output (O) of each type of virus. Figure 3. Electron micrograph of negatively stained EAMs exhibiting a gross structure and size identical to that of adenovirus. Figure 4. Southern blot analysis of DNA prepared from Ad5[beta]PDE EAMs with probes from the C-terminus of [beta]-PDE (P) or the N-terminus of LacZ (L). Note the presence of monomers (M), head (H)-to-tail (T) dimers, H-to-H dimers and T-to-T dimers. The orientation of the adenoviral packaging signals ([Psi]), [beta]-PDE cDNA ([beta]), pBSIIKS (O) and LacZ gene with respect to the adenovirus left and right inverted terminal repeats (LITR/RITR) in each dimer is shown. A total of 3.4 × 107 purified EAMs (Ad5[beta]PDE) were injected into the subretinal space of homozygous rd mice by a trans-choroidal approach between 5 and 6 days postnatal, a time-point before the onset of retinal degeneration. The contralateral eye of the same mouse was injected with Ad5[beta]N-an EAM identical in all respects to Ad5[beta]PDE except that it does not contain a [beta]-PDE cDNA. To determine whether [beta]-PDE was expressed in Ad5[beta]PDE-transduced retina, we examined retinas from injected eyes for production of [beta]-PDE transcripts and for cGMP-PDE activity. Total RNA was extracted from Ad5[beta]PDE and Ad5[beta]N-injected retinas at 2 week intervals until 18 weeks post-injection (the latest time point examined), reverse transcribed and subjected to PCR. RT-PCR amplification resulted in a fragment of 650 bp only in RNA templates prepared from retinas injected with Ad5[beta]PDE. Since the rd mouse has an ochre mutation in codon 347, converting TAC to TAA, a new DdeI site is created in rd homozygotes (17). Incubation of the RT-PCR products with DdeI followed by fractionation on agarose gels identified digestion products associated only with the transduced gene (Fig. Figure 5. RT-PCR amplification of transgene and native [beta]-PDE transcripts from Ad5[beta]PDE transduced rd retinas. PCR amplification from cDNA using [beta]-PDE specific primers RTF and RTR results in a 650 bp product encompassing exon 7 of the murine [beta]-PDE gene. Homozygous rd mice have been shown previously to contain a DdeI restriction site at nt 1045 in addition to the one at nt 907. RNA templates arising from Ad5[beta]PDE-transduced cells only have one DdeI site at position 907. RT-PCR was performed from retinal cDNA prepared from injected mice at various ages post-injection in 2 week intervals up to 18 weeks. RNA was either treated with (+) or without (-) reverse transcriptase prior to PCR. We have qualitatively examined expression of the [alpha] and [beta] subunits of PDE by western analysis in Ad5[beta]PDE and Ad5[beta]N-injected rd retinas up to 147 days postnatal. The [alpha] subunit of PDE could barely be detected at day 19 postnatal in the Ad5[beta]N-injected rd retina. Both the [alpha] and [beta] subunits of PDE could be detected up to 90 days postnatal in Ad5[beta]PDE-injected rd retina (Fig. Figure 6. Western analysis of [alpha]-PDE and [beta]-PDE subunits in Ad5[beta]PDE- (+) or Ad5[beta]N-injected (-) rd/rd retinas through day 147 postnatal. Untreated aged-matched C57BL (+/+) retinas at days 12, 33 and 119 were used as positive controls. Note that this is an amplified signal and hence not a representation of the stoichiometric ratio of each subunit. We have also measured the activity of cGMP-PDE in Ad5[beta]PDE or Ad5[beta]N-injected rd retinas up to 147 days post-injection. Our results indicate that the [beta] subunit of cyclic GMP PDE is processed and forms a functional PDE complex with the [alpha] and [gamma] subunits. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal in Ad5[beta]PDE injected rd retina (Fig. It has been shown previously that the light-activated state of PDE can be mimicked by treating the preformed PDE [alpha][beta][gamma][gamma] complex with trypsin, histone, or polycations, such as protamine, which selectively digest or remove the PDE[gamma] subunit inhibitory constraint (36). The kinetics of PDE activation in vitro correspond to the degradation of PDE[gamma] by trypsin (37). Homogenates were prepared from control retinas and from three Ad5[beta]PDE- or Ad5[beta]N-injected rd/rd retinas at 12 days postnatal and assayed for PDE activity after treatment with trypsin. Trypsinization markedly increased PDE activity in wild-type mice (22-fold) and Ad5[beta]PDE-injected mice (10-fold) but minimally in Ad5[beta]N-injected or rd homozygotes (Fig. Outer nuclear layer (ONL) thickness is considered a reliable index for comparing photoreceptor cell number between retinas subjected to various traumatic or genetic insults (38). To obtain a quantitative estimate of the possible differences in the number of photoreceptors harbored by our experimental retinas, representative sections taken at different intervals post-injection were scored for ONL thickness. Whereas there are eight to 10 rows of photoreceptor nuclei in mature wild-type mice or heterozygous rd mice, there is only a single row of nuclei in homozygous rd mice at 17 days postnatal. All photoreceptor nuclei are absent in the rd retina by 6 weeks postnatal. Examination of ONL thickness at 2 week intervals in Ad5[beta]PDE or Ad5[beta]N-injected rd retinas up to 16 weeks post-injection (the latest time point examined) still showed significant differences up to 12 weeks post-injection(Figs Figure 7. Actual and mean cGMP-PDE activity in rd/rd retinas after treatment with Ad5[beta]PDE and Ad5[beta]N. Activities were measured at days 12, 19, 33, 63, 91, 119 and 147 postnatal (n = 3-5 retinas). Data from all retinas are included in the above figure; however, it is unlikely that the two samples indicated by (*) were injected in the subretinal space (see text). Incorporation of the E.coli Lac Z gene in our construct has enabled us to determine the total spread of EAMs across the subretinal space. Our data indicate that an area of ~1 mm2 surrounding the injection site is infected by a single EAM injection (Fig. Finally, although our results are promising, we are attempting to improve the viral system employed in this study further. As there is some contamination of helper virus in our system (albeit with a first generation E1/E3-deleted virus) it is possible that a slow immune response may be generated against virally transduced cells. It is not known whether an immune response might be generated against the [beta]-PDE transgene or [beta]-galactosidase, also possibly resulting in immune-mediated arrest of rescue. We are currently carrying out detailed experiments to answer some of these questions. In addition, the emergence of defective virions allows co-migration of the defectives and EAMs in the CsCl preparations. We are presently testing materials such as rubidium chloride and potassium bromide which have higher resolving power than CsCl at [rho] = 1.3 (39) in order to obtain purer stocks of EAMs. In summary, we have shown that transgene products can be detected in EAM-injected rd retinas for at least 18 weeks post-injection; the [alpha] and [beta] subunits of PDE could be detected up to 90 days postnatal with a maximal PDE activity of 150 nm/min/mg at 33 days postnatal. The outer nuclear layer of rd retinas was rescued for at least 12 weeks post-injection. These results demonstrate an improved level of rescue over the use of first-generation adenoviral vectors and contribute significantly towards the long-term goal of finding a therapy for some retinal and CNS disorders.
MATERIALS AND METHODS
Construction of pAd5[beta]PDE, pAd5[beta][Mgr] and purification of EAMs
Cloning was carried out using standard techniques and as described previously (7). The murine [beta]-PDE cDNA was obtained as a 2.75 EcoRI fragment from pBB1 (40). This cDNA was cloned downstream of a 350 bp human [beta]-PDE promoter (41). The inverted Ad origin of replication and five encapsidation signals were derived from pFG140 (42). The resulting plasmid (Fig.
Figure 8. Cyclic GMP-PDE activity following treatment with (+) or without (-) trypsin in Ad5[beta]N or Ad5[beta]PDE injected rd/rd retinas at 12 days postnatal. Age-matched C57BL mice were used as positive controls. Figure 9. Light micrograph (630×) of Ad5[beta]PDE-injected (C1-C6; 2-12 weeks post-injection in 2 week intervals) or Ad5[beta]N-injected (B1-B4, at 2, 4, 6 and 12 weeks post-injection, respectively) retinas. The outer layers of the adult retina from a C57BL mouse (A) are presented as a control. OS, outer segments; IS, inner segments; OPL, outer plexiform layer; ONL, outer nuclear layer; IPL, inner plexiform layer; INL, inner nuclear layer. Note that there appears to be no cellular infiltration in the injected retinas. Fractions of lysate prepared during various serial passages were used to determine the total titer of the EAM and helper virus prior to subretinal injection. As the EAM and helper virus encode an E.coli Lac Z gene and human placental alkaline phosphatase gene, respectively, we performed [beta] galactosidase and alkaline phosphatase assays on infected 293 cells to determine the total titer of the two types of virions. Infected 293 cells were assayed as described previously (7). Approximately 2 × 109 Ad5[beta]PDE EAMs were incubated in 0.1 M Tris, pH 8, 2 mM EDTA, 1% SDS, 1 mg/ml Pronase, at 37°C overnight and extracted with phenol-chloroform. A total of 3 µg of viral DNA were digested with NcoI, fractionated on a 1% agarose gel, electroblotted onto a nylon membrane (Hybond-N) and hybridized either with a 32P-labeled restriction fragment from the C-terminus of the [beta]-PDE (NcoI-Bst1107I fragment from pAd5[beta]PDE) cDNA or the N-terminus of the E.coli Lac Z gene (NotI-BssHII fragment from pAd5[beta]PDE). All animals were cared for in accordance with the Association for Research in Vision and Ophthalmology `Statement for use of animals in ophthalmic and vision research' and in compliance with federal, state and local regulations. C57BL (control) and C57BL rdle/rdle mice were bred in our vivarium and reared under 12 h daily cycles of light and darkness. Mice were anesthesized with i.p. administration of 2,2,2-tribromoethanol (0.5 g/kg) between 5 and 6 days postnatal. Injections were performed with a trans-scleral, trans-choroidal approach (31) using a 33 gauge needle (15° bevel) with a 5 µl Hamilton syringe mounted on a micromanipulator. A total of 3.4 × 107 purified Ad5[beta]PDE EAMs (or Ad5[beta]N EAMs in the contralateral eye) in 1.0-1.5 µl PBS was delivered at a rate of 0.5 µl/min. Figure 10. Comparison of outer nuclear layer column height in terms of nuclei in Ad5[beta]PDE- or Ad5[beta]N-injected rd/rd retina in 2 week intervals up to 16 weeks post-injection (117 days postnatal). PDE activity analyses. PDE activity was measured essentially as described previously (46) using a two-step procedure. Briefly, samples (2-5 µg protein) were incubated at 37°C for 15 min with 250 µM [3H]cGMP, followed by boiling. After cooling, samples were incubated with 0.4 U alkaline phosphatase at 37°C for 10 min prior to the addition of 1 ml of 1:3 slurry of AG1-X2 resin (Bio-Rad). Samples were left standing at 4°C for 30 min, centrifuged at 3500 g for 20 min, and supernatants were counted. In some experiments PDE was trypsin activated.
EAM and helper virus titers
Genomic structure of Ad5[beta]PDE
Administration of EAMs
Analyses of [beta]-PDE expression
RT-PCR. Mice were sacrificed by cervical dislocation and the retinas were dissected rapidly after enucleation. RNA was extracted from Ad5[beta]PDE- or Ad5[beta]N-injected rd retinas at 1, 2, 4, 6, 8, 12 and 18 weeks post-injection using TRIzol (Gibco BRL) according to the manufacturer's instructions, and PCR-amplified with or without prior RT. The RT reaction was carried out in PCR buffer (1 mM each dNTP, 500 mM KCl, 200 mM Tris-HCl, pH 8.4, 25 mM MgCl2) with 1 U/µl RNase inhibitor (Perkin Elmer), 100 pmol random hexamers (Amersham), 1 µg total RNA, 1 mg/ml nuclease free BSA and 100 U MuLV reverse transcriptase in a final volume of 20 µl. The reaction was incubated at 23°C for 10 min and 42°C for 30 min. The RNA-cDNA hybrid was denatured, and reverse transcriptase was inactivated at 95°C for 10 min prior to quick-chill on ice. For cDNA amplification, the RT reaction was brought up to 100 µl in 1× PCR buffer with 100 ng each oligonucleotide primer (5[prime]-CCACAACTGTGAGACACGCAG-3[prime] and 5[prime]-TACGGTTCTCCAGCTTGTTCATC-3[prime]) and 3 U of Taq polymerase. PCR cycles were performed in a Stratagene robocycler with an initial denaturation at 94°C for 4 min followed by 40 cycles at 94, 55 and 72°C for 1 min each. PCR products were extracted with phenol-chloroform (1:1) and digested with DdeI prior to fractionation on agarose gels.
Histology and [beta]-galactosidase expression
Eyes were enucleated and retinas fixed overnight in 1% formaldehyde and 2% glutaraldehyde. Retinas were immersed in 1% osmium tetroxide for 1 h and dehydrated through a graded series of alcohol. Tissue (2 mm2 sections) was embedded in araldite and propylene oxide and sectioned at 0.5 µm. Single sections of the retina (extending from the superior ora serrata to the inferior ora serrata) traversing the injection site (and some randomly selected regions of the retina) were studied by light microscopy. Assessment of photoreceptor nuclei was performed in a scheme described previously (28). For analyzing [beta]-galactosidase expression, whole eyes were punctured, washed in PBS and fixed overnight in 0.5% glutaraldehyde. Eyes were washed in PBS and reacted with X-gal (5-bromo-4-chloro-3-indolyl-[beta]-d-galactosidase) at 1.2 mg/ml in PBS containing 10 mM each of K3Fe(CN)6 and K4Fe(CN)6-3H2O, and 2 mM MgCl2.
Detection of wild-type virus
As only one recombination event between the hpAP virus and E1 sequences in the 293 cells is required to obtain a replication competent adenovirus (RCA), we evaluated all stocks of EAMs for contamination with RCA by infectivity assay on HeLa cells at a multiplicity of infection of 10 (infection for 12 days). No RCAs were detected throughout our studies.
Figure 11. [beta]-Galactosidase staining in Ad5[beta]PDE-injected (A) or PBS-injected (B) eyes from a BALB/c mouse at 3 weeks post injection. Note the intense staining covering an area of ~1 mm2 indicated by the arrow. We would like to thank Ken Tran, Clyde Yamashita, Tammy Rickabaugh and Elliot Brill for excellent technical assistance. This study was supported by grants from the Foundation Fighting Blindness, the National Institutes of Health (EY08285 and EY02651) and the Chatlos Foundation. D.B.F. is recipient of a Senior Investigator Award from Research to Prevent Blindness. R.K.-S. is recipient of a Young Investigator Award from the Foundation Fighting Blindness.
ACKNOWLEDGEMENTS
REFERENCES
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