Lacrimal Gland as a Target Organ for Adenovirus Gene Therapy Encoding Erythropoietin for Dry Eye Induced by Benzalkonium Chloride

ABSTRACT

Purpose: The aims of this work were a) to describe the histology of the lacrimal gland (LG) and cornea induced by an adenovirus (Ad) vector encoding the human erythropoietin (Epo) gene delivered to the LG and b) to evaluate the therapeutic potential of this strategy to prevent benzalkonium chloride (BAK) corneal toxicity.

Methods: Structure and function of male Wistar rats LG were compared in the groups: 1) naïve control and 2) Ad-hEpo in the right LG (RLG). The protective response against BAK eye drops was compared among the groups 1) naïve control, 2) BAK in the right eye, 3) Ad-hEpo RLG + BAK and 4) Ad-hEpo in the right salivary gland (RSG)+BAK. Ad-hEpo groups received an injection of AdLTR2EF1a-hEPO (25 ul, 10¹⁰ particles/ml) in the right LG or SG (positive control). The BAK groups received 0.2% BAK in the right cornea twice a day. The tests applied after 7 days, included tear secretion, hEPO mRNA detection by qRT-PCR, LG and cornea histology, LG ELISA for cytokines and hematocrit.

Results: hEPO mRNA was present in the Ad-hEpo RLG and RSG, but not kidney or liver samples (negative controls). TNF-α and IL-1β increased in the LG exposed to Ad-hEpo compared to naïve control (p = .0115 and p = .0397, respectively). BAK reduced tear secretion, but this reduction was prevented by Ad-hEpo RLG+BAK and Ad-hEpo RSG+BAK (p = .017). The corneal epithelia were thinner in the BAK-treated groups independent of Ad-hEpo (p = .0009). Hematocrit increased only in the Ad-hEpo RSG group (p = .01).

Conclusions: Ad-hEpo infection of rat LG and SG induces local, but only the SG infection induced systemic changes in rats. Importantly, Ad-hEpo attenuated the BAK-mediated toxic reduction in tear flow. Future studies must consider viral vector tissue tropism, biodistribution and effective therapeutic gene products for ocular surface diseases.

KEYWORDS:

• Adenovirus
• cornea
• erythropoietin
• lacrimal gland
• salivary gland
• viral vector

Acknowledgments

The study was supported by grants from the following Brazilian governmental institutions. The authors would like to thank the support by grants from the following Brazilian governmental institutions: Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) (nº 2015/20580-7 and 2014/22451-7) (São Paulo, SP, Brazil); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (n^o: 474450/2012-0) (Brasilia, DF, Brazil); CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) (Finance Code 001) (Brasilia, DF, Brazil); Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clinicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA) (669/2018) (Ribeirão Preto, SP. Brazil); and Research Core of Ocular Physiopathology and Therapeutics from University of São Paulo (NAP-FTO) (nº 12.1.25431.01.7) (Ribeirão Preto, SP. Brazil).

Disclosure statement

No competing financial interests exist.

Additional information

Funding

This work was supported by the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) [Finance Code 001]; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Brasilia, DF, Brazil [no: 474450/2012-0]; Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clinicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA), Ribeirão Preto, SP. Brazil [669/2018]; Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) (São Paulo, SP, Brazil) [nº 2015/20580-7 and 2014/22451-7]; Research Core of Ocular Physiopathology and Therapeutics from University of São Paulo (NAP-FTO) Ribeirão Preto, SP. Brazil [nº 12.1.25431.01.7].