Adenovirus-mediated gene transfer to human lens epithelial cells in organ culture

laboratory science

Adenovirus-mediated gene transfer to human lens epithelial cells in organ culture Louis M. Carrington, MPhil, Tom Southgate, BSc, Lisa A. Saxby, PhD, Khaled Abul-Hassan, MSc, Tricia C. Maleniak, BSc, Maria G. Castro, PhD, Michael E. Boulton, PhD ABSTRACT Purpose: To assess the feasibility of using recombinant adenovirus vectors to transduce the human lens epithelial cells (LECs) involved in posterior capsule opacification (PCO). Setting: Department of Ophthalmology and Molecular Medicine Unit, University of Manchester, Manchester, United Kingdom. Methods: Seventeen human lens capsules were maintained in organ culture to allow LECs to proliferate onto the posterior capsule. Partly covered and completely covered capsules were infected with a recombinant adenovirus vector RAd35, encoding for the marker gene ␤-galactosidase at plaque-forming units per milliliter (pfu/mL) ranging from 107 to 1010 for up to 48 hours. Assessment of infection and transduction of the marker gene were achieved by calculating the percentage of cells exhibiting X-gal staining both macroscopically and microscopically. Results: Staining appeared to be dependent on virus dose, with most intense staining at doses of 108 and 109 pfu/mL with decreased staining at higher and lower viral doses. Microscopic assessment demonstrated that all cells expressed ␤-galactosidase when infected with 109 pfu, 84% at 108 pfu, and 45% at 107 pfu. At 1010 pfu, some cytotoxicity was observed. Conclusions: These results indicate that recombinant adenoviruses can be used to transfer genes to the LECs involved in PCO. The transfer of cytotoxic genes after cataract surgery may be considered a preventive measure for PCO. J Cataract Refract Surg 2000; 26:887– 892 © 2000 ASCRS and ESCRS

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osterior capsule opacification (PCO) is a common complication of cataract surgery that affects up to 50% of patients within 2 months to 5 years after extraAccepted for publication November 24, 1999. Reprint requests to Dr. Michael Boulton, Cellular and Molecular Biology Unit, Department of Optometry and Vision Sciences, Cardiff University, PO Box 905, Cardiff CF1 3XF, United Kingdom. E-mail: [email protected]. © 2000 ASCRS and ESCRS Published by Elsevier Science Inc.

capsular cataract extraction (ECCE).1,2 The incidence of PCO is far greater in patients with congenital cataract than in those with senile cataract and is thought to reflect the higher proliferative capacity of lens epithelial cells (LECs) in the young.3 In PCO, residual LECs in the anterior and equatorial regions of the lens capsule after surgery proliferate and migrate onto the previously acellular posterior capsule. These cells form a multilayer, take on a fibroblast0886-3350/00/$–see front matter PII S0886-3350(00)00325-4

LABORATORY SCIENCE: LENS EPITHELIAL CELL GENE TRANSFER

like appearance, contract, and secrete extracellular matrix.1,4 The resulting fibrocellular membrane causes significant visual loss when it reaches the visual axis, necessitating a posterior capsulotomy, which is also associated with several ocular complications.1,4 Many approaches have been used in an attempt to prevent PCO. These include trying to remove all LECs during surgery,5,6 applying pharmacological agents such as antimitotics and toxins,7,8 and modifying the intraocular lens implanted in the capsular bag at the time of surgery.9 –11 Although these interventions have shown promise in vitro or in animal models, they have had limited success when used in patients. An alternative approach is to use gene therapy to eliminate the proliferating cells, preventing PCO. The transfer of direct and conditional cytotoxic genes in vitro and in vivo, mainly used for cancer treatment, is well documented.12–16 Although this approach offers a more selective alternative for eliminating the cells involved in PCO, the potential use of gene therapy to deliver genes to these cells has not been investigated. This study, therefore, assessed the feasibility of using recombinant adenovirus vectors to transduce the human LECs involved in PCO.

Materials and Methods Recombinant Adenoviral Vector Construction and Purification Construction and characterization of RAd35 has been reported17,18 and grown and purified as described (Figure 1).14,19 Briefly, the transfer vector pAL119/lacZ was constructed from pXCX2 with addition of a linker containing the HindIII cloning site at the XbaI cleavage site. The lacZ was cloned under the control of the MIEhCMV promoter and upstream of a polyadenlylation signal on a HindIII expression cassette. It was then cotransfected with pJM17 (Microbix Biosystems Inc.) into HEK-293 cells by calcium phosphate co-precipitation. Homologous recombination resulted in the recombinant adenovirus RAd35. The virus was propagated on HEK-293 cells, purified twice on prepared cesium chloride gradients, and dialyzed twice against a buffer of 10 mM Tris, 1 mM magnesium chloride, 135 mM sodium chloride pH7.5, and once against the same buffer containing glycerol 10%. The virus was titrated by plaque assay on HEK-293 cells, and the viral titer was determined to be 1 ⫻ 1012 plaque-forming units per milliliter (pfu/mL). Levels of

Figure 1. (Carrington) Schematic of the recombinant adenovirus expressing lacZ under the control of a short human major immediate early promoter of cytomegalovirus. The cDNA encoding lacZ was cloned into plasmid pAL119. Plasmid pAL119/ lacZ thus contains the promoter, lacZ, followed by a polyA signal. The transgenic sequences are flanked by adenoviral sequences, which recombine up and downstream of the pBRX insert in pJM17 to originate the recombinant replication deficient adenovirus containing the transgenic sequences.

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endotoxin were measured using the E-TOXATE威 assay (Sigma), based on the limulus amebocyte lysate clotting reaction and were determined to be negative based on results being below 2.0 endotoxin units/mL.20 Levels of replication-competent adenovirus in the viral preparation used was assessed by the method of Dion and coauthors21 and found to be negative. Organ Culture Model Seventeen human lens capsules were prepared from eyes donated for research as described.22 In brief, the anterior segment was dissected away, exposing the lens. Part of the lens capsule was removed by an anterior capsulorhexis, and the cortex and nucleus were expressed by hydrodissection. After a sterile silicone ring was inserted into each capsule to maintain structure, the capsule was detached and transferred to the well of a 6-well plate containing serum-free Waymouth’s MB 752/Ham’s F12 medium supplemented with nonessential amino acids (1% ⫻100), antibiotics (200 ␮g/mL kanamycin, 120 ␮g/mL benzylpenicillin, 290 ␮g/mL streptomycin sulphate), and L-glutamine (200 ␮g/mL). Capsules were incubated for different durations to yield 2 groups of specimens: those in which the anterior surface of the posterior capsule was partly covered by LECs and those that were completely covered. Qualitative Assessment of Cell Staining as a Function of Virus Dose Ten human lens capsules were infected with increasing doses of RAd35 ranging from 107 to 1010 pfu in 250 ␮L of serum-free Waymouth’s MB 752/Ham’s F12 medium as described. After 48 hours, the capsules were briefly washed in Dulbecco’s phosphate buffered solution (PBS) and fixed for 1 hour with paraformaldehyde 4% containing 0.12 M sucrose dissolved in PBS. They were incubated for 3 hours at 37°C in a solution containing 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, and 1 mg/mL of chromogen X-gal (Sigma Co.). The distribution and degree of staining for each capsule were assessed macroscopically, and the ratio of infected cells (stained blue) versus uninfected cells was determined by examination under an inverted microscope. Expression of ␤-galactosidase is directly related to the infectability of the cells, which means the more infectable the cells, the greater the ␤-gal expression. Capsules were subsequently dehydrated through a

graded series of alcohols and immersed in xylene before being embedded in paraffin wax. Seven micron thick sections were used for microscopic examination and assessment of transduced cells. Assessment of Recombinant Adenoviral Gene Transfer as a Function of Time Seven human lens capsules were infected in 250 ␮L of serum-free Waymouth’s MB 752/Ham’s F12 medium, as described above, containing 1 ⫻ 108 pfu of RAd35 in each well. At 3, 6, 12, 24, 33, and 48 hours, the capsules were briefly washed with Dulbecco’s PBS and then fixed and stained as above.

Results Expression of ␤-galactosidase in LECs as a Function of Recombinant Adenovirus Dose Gross anatomy revealed successful adenovirus mediated gene transfer into human LECs in organ culture. Positive X-gal staining was apparent throughout the cellular regions of the pre- and post-covered capsules (Figure 2, A). No staining was observed in the acellular region of the precovered capsule or the noninfected control specimens. Staining appeared to be dependent on the viral input, with most intense staining at doses of 108 and 109 pfu. Decreased X-gal staining was observed at higher and lower viral doses. Examination by inverted microscopy confirmed the gross anatomical appearance (Figure 2, B). All cells appeared to be stained at 109 pfu, but the number of positively stained cells decreased with decreasing virus dose: 84% and 45% at 108 and 107 pfu, respectively. At the highest virus dose used, 1010 pfu, cells had detached from the capsule, possibly as a result of virus cytotoxicity caused by the high viral input. Histological analysis of the sections confirmed that cells were stained throughout all the layers on the capsule (Figure 2, C). Expression of ␤-galactosidase as a Function of Length of Exposure to Recombinant Adenovirus Gross anatomy and examination by inverted microscopy revealed that optimal infection was achieved after 33 hours of virus exposure. At earlier times, not all cells were expressing ␤-galactosidase, and at longer exposures (48 hours) cell loss was observed.

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Discussion

Figure 2. (Carrington) Gross anatomy and light microscopy study after infection of human LECs on the posterior lens capsule using the recombinant adenovirus RAd35 expressing ␤-galactosidase. Appearance of lens capsules after infection with 108, 109, and 1010 pfu/mL of RAd35 (A). No blue stain was observed in the noninfected control (0) or in the acellular center of a partially covered capsule (108) (original magnification ⫻1.7). Inverted microscopy appearance of infected human lens cells on a partially covered posterior capsule infected with 109 pfu (original magnification ⫻170) (B). Prepared sections (7 ␮m) showed that the X-gal stain was located within the LECs (C) (original magnification ⫻260). Note that the cell layer (thick arrow) has detached from the capsule (thin arrow). 890

Ours is the first study to demonstrate gene transfer into the human LECs involved in PCO. The introduction of tissue-specific, conditionally or directly cytotoxic genes into (1) LECs remaining at the time of ECCE extraction or (2) LECs as they cover the posterior capsule offers the potential to kill the cells responsible for the subsequent development of PCO. For example, one could use the prodrug activating enzyme thymidine kinase from HSV1, which converts nucleoside analogs (e.g., acyclovir or ganciclovir) into their phosphorylated metabolites. The metabolites then act as competitive inhibitors of endogenous nucleotides for incorporation into replicating DNA of proliferating cells, causing cell death. Another prodrug activating enzyme that could be used is cytosine deaminase, which activates 5-fluorocytosine to the cytotoxic agent 5-fluorouracil. Directly cytotoxic genes could include bacterial toxins; for example, Pseudomonas exotoxin type A, which causes cell death by inhibiting protein synthesis. Using adenoviral vectors, we have shown it is possible to restrict the expression of the therapeutic transgene with cell-type specific promoters,14,17 limiting the cytotoxicity to the desired cell-type/tissue. We therefore envisage that by combining transcriptional targeting with cytotoxic gene delivery, it will be possible to develop a feasible gene therapy strategy for PCO. Gene therapy has been shown as a feasible approach in the treatment of several ocular disorders. In vivo, viral gene transfer has been successfully achieved in many ocular structures, including the cornea, lens, ciliary body, and retina, with minimal resulting immunogenicity.23–27 Such gene transfer has successfully delayed the death of photoreceptors in inherited retinal degeneration,24,25 rescued injured retinal ganglion cells,27 restored lysosomal function in mucopolysaccharidosis type VII,28 and retarded the progression of proliferative vitreoretinopathy by targeting proliferating cells with suicide genes.29 Furthermore, ex vivo infection of corneal endothelium before transplantation may prove to prevent allograft rejection.30,31 Further studies are essential to confirm the efficacy of gene-directed cytotoxicity to prevent PCO in vivo. However, this approach could be used in conjunction with the transfer of directly or conditionally cytotoxic genes to eliminate the remnant LECs responsible for the

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development of PCO. This approach is amenable to use during surgery as successful gene transfer has been observed in other tissues after short-term exposure to adenoviruses.32,33 Site specificity can be achieved by incorporating a lens-specific promotor into the construct. A successful outcome will lead to clinical trials with a view to reducing the occurrence of PCO, which affects up to 50% of patients who have had cataract surgery.

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From the Department of Ophthalmology (Carrington, Saxby, AbulHassan, Boulton) and Molecular Medicine Unit (Southgate, Maleniak, Castro), University of Manchester, Manchester, United Kingdom. Funded by Research into Ageing and the Iris Fund. Development of gene therapy strategies was supported by Action Research, the MRC (UK), BBSRC, The Wellcome Trust, CRC (UK), The Parkinson’s Disease Society, REMEDI, Sir Halley Stewart Trust, and The Royal Society. None of the authors has a financial or proprietary interest in any product or material mentioned.

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