Gene transfer to the outflow tract

Abstract

Elevated intraocular pressure is the primary cause of open angle glaucoma. Outflow resistance exists within the trabecular meshwork but also at the level of Schlemm's canal and further downstream within the outflow system. Viral vectors allow to take advantage of naturally evolved, highly efficient mechanisms of gene transfer, a process that is termed transduction. They can be produced at biosafety level 2 in the lab using protocols that have evolved considerably over the last 15-20 years. Applied by an intracameral bolus, vectors follow conventional as well as uveoscleral outflow pathways. They may affect other structures in the anterior chamber depending on their transduction kinetics which can vary among species when using the same vector. Not all vectors can express long-term, a desirable feature to address the chronicity of glaucoma. Vectors that integrate into the genome of the target cell can achieve transgene function for the life of the transduced cell but are mutagenic by definition. The most prominent long-term expressing vector systems are based on lentiviruses that are derived from HIV, FIV, or EIAV. Safety considerations make non-primate lentiviral vector systems easier to work with as they are not derived from human pathogens. Non-integrating vectors are subject to degradation and attritional dilution during cell division. Lentiviral vectors have to integrate in order to express while adeno-associated viral vectors (AAV) often persist as intracellular concatemers but may also integrate. Adeno- and herpes viral vectors do not integrate and earlier generation systems might be relatively immunogenic. Nonviral methods of gene transfer are termed transfection with few restrictions of transgene size and type but often a much less efficient gene transfer that is also short-lived. Traditional gene transfer delivers exons while some vectors (lentiviral, herpes and adenoviral) allow transfer of entire genes that include introns. Recent insights have highlighted the role of non-coding RNA, most prominently, siRNA, miRNA and lncRNA. SiRNA is highly specific, miRNA is less specific, while lncRNA uses highly complex mechanisms that involve secondary structures and intergenic, intronic, overlapping, antisense, and bidirectional location. Several promising preclinical studies have targeted the RhoA or the prostaglandin pathway or modified the extracellular matrix. TGF-β and glaucoma myocilin mutants have been transduced to elevate the intraocular pressure in glaucoma models. Cell based therapies have started to show first promise. Past approaches have focused on the trabecular meshwork and the inner wall of Schlemm's canal while new strategies are concerned with modification of outflow tract elements that are downstream of the trabecular meshwork.

Keywords: Gene therapy; Glaucoma; Intraocular pressure; Outflow tract; Trabecular meshwork.

Fig. 1. Genome and structure models of vectors commonly used in ocular gene transfer

Adenoviral vectors are non-integrating and can deliver up to 38 kilo-basepairs (kb). Adeno-associated viral vectors may integrate or persist as concatemers and have a small physical size with a packaging capacity of 1.6 kb. Herpes simplex viral vectors do not integrate and have a very large capacity of up to 150 kb. Newer adeno- and herpes viral vectors are less immunogenic than earlier generations. Retroviral vectors have a packaging capacity of 7 kb and only transduce dividing cells into which they permanently integrate. Lentiviruses belong to the family of retroviridae but have evolved a nuclear import mechanism for nondividing cells. Derived vectors have a capacity of 7 kb and permanently integrate into both dividing and nondividing cells. All vectors shown here, but not the adeno-associated and retroviral vector, are capable of delivering complex genes that include introns. All of them can deliver exons and small, non-coding RNA (siRNA).

Fig. 2. Anterior segment perfusion culture system

(Loewen et al., 2016) (A). FIV transduction of human (B) and porcine eyes (C). A redesigned culture bottom allows direct observation of vector function through the bottom of the dish (C).

Fig. 3. Live observation of transgene expression with a standard goniolens

Setup for observation of mid-sized animals (A). Serial observation of EGFP marker gene expression in a macaque eye following an intracameral FIV bolus (B (Barraza et al., 2009)). Relatively selective TM transduction and marker gene in the domestic cat (C (Loewen et al., 2004a)).

Fig. 4. Examples of gene therapeutic IOP lowering

Prostaglandin pathway transduction shows a long-term IOP reduction after a single vector bolus in the cat (A (Barraza et al., 2010)). Inducible cytoablative vector causes a rapid TM cell loss followed by a recovery of IOP and TM cell numbers (Zhang et al., 2014) in this model of TM regeneration (B).

Fig. 5. New cell and gene delivery approaches

(A) Adipose derived tissue stem cells transduced with EGFP expressing FIV vector (Zhang et al., 2014) were seeded into the TM of anterior perfusion cultures of human eyes. View from inside into everted eye. (B) Magnified view of TM from experiment (A). (C) Collector channel (marked by red arrowheads) transduction with EGFP expressing FIV vector. External, frontal view of perfused porcine eyes (Loewen et al., 2016). An ab interno trabeculectomy (Kaplowitz et al., 2014) was performed followed by an intracameral vector bolus. (D) Cell seeding model with EGFP expressing fibroblasts (Oatts et al., 2013). External, frontal view of perfused porcine eyes. An ab interno trabeculectomy (Kaplowitz et al., 2014) was performed followed by an intracameral cell bolus. Collect channels were marked by red arrowheads.