Gene delivery by viral vectors in primary cultures of lacrimal gland tissue.
OBJECTIVE
To test the feasibility of gene transfer into lacrimal gland tissue in primary culture, using different viral vectors.


METHODS
Lacrimal glands were dissected from adult Sabra rats and divided by pincers to 0.3-0.4 mm fragments. Tissue was maintained under primary organ culture conditions using the "raft" technique. The ability of three different viral vectors to conduct beta-galactosidase (beta-gal) gene delivery was examined: adenovirus (Ad5CMVLacZ), vaccinia (VSC9), and herpesvirus (tkLTRZ(1)). Tissue fragments were incubated for 60 minutes with one of the viral vectors and transferred to fresh medium. After 3 and 7 days, beta-gal expression was examined by X-gal staining in gross preparations and in histologic sections.


RESULTS
At 3 days, beta-gal expression was observed in 33% of tissue fragments exposed to the vaccinia vector and in 18% and 14% of fragments exposed to the adenoviral and herpes vectors, respectively. After 7 days in culture, successful gene delivery occurred in 77% of vaccinia, 41% of adenovirus, and only 13% of herpesvirus applications. Vector-specific reporter gene expression patterns were observed: With the vaccinia vector, lacrimal duct cells were predominantly stained; in contrast, the adenoviral vector tended to transduce the interacinar areas, with beta-gal expression mainly occurring within the myoepithelial cells.


CONCLUSIONS
Vaccinia and adenovirus are efficient vectors for gene transfer into lacrimal gland tissue in primary culture. The specific expression pattern obtained by the vaccinia vector probably reflects its characteristic tissue tropism to lacrimal duct cells. The results presented in this ex vivo system may be a first step toward expressing genes with products that could be continuously delivered to the eye through the tears. Such proteins could include anti-inflammatory, anti-angiogenic, anti-herpetic, anti-bacterial, or anti-glaucomatous agents, among others.
Banin
Eyal
E
Department of Ophthalmology, The Hebrew University-Hadassah Hospital and Medical School, Jerusalem, Israel. banine@md.huji.ac.il
Obolensky
Alexey
A
Piontek
Elena
E
Falk
Haya
H
Pikarsky
Eli
E
Pe'er
Jacob
J
Panet
Amos
A
Chowers
Itay
I
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D005697:Galactosides / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D007211:Indoles / Q000378:metabolism; D007765:Lacrimal Apparatus / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D009924:Organ Culture Techniques; D011817:Rabbits; D018139:Simplexvirus / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics*
D005697:Galactosides; D007211:Indoles; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside