Rational for Developing Gene Transfer to the Trabecular Meshwork

In the presence o f poor long-term outcome o f vision in glaucoma patients with current strategies gene therapy may offer solutions to permanently correct aqueous humor outflow. Lentiviral vectors have evolved as the only vector type that allows stable and targeted genetic modification o f the trabecular meshwork providing new tools for development o f glaucoma therapy and animal models through tissue specific transgenesis. Permanent genetic reprogramming o f anterior chamber outflow tract physiology by gene therapy has attracted attention as an ideal solution in theory because o f the disease’s life-long chronicity and the emerging understanding o f its genetic b a s i s . T h e trabecular meshwork (TM), is a key intraocular structure to target since it controls lOP by controlling outflow o f aqueous humor.

Strategies for gene therapy for glaucoma must not only consider the chronic nature o f the disease but also the generally nondividing nature o f the target cells. Two general approaches have emerged. One focuses on the intraocular pressure problem, and the other on blocking its sequelae, retinal ganglion cell death.^^^’ The accessibility o f the anterior chamber and the restricted anatomic target are favorable for gene therapy

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directed at the TM. Gene therapy with retroviral vectors has appeal, because these vectors undergo reverse transcription o f their single-stranded RNA genomes, generating a linear double-stranded DNA intermediate that that is subsequently integrated into the host genome in a reaction catalyzed by the retroviral integrase.

Therefore, these vectors result in permanent transgenes and have potential to address the chronicity o f glaucoma pathophysiology. However, an important consideration is that TM cells do not normally divide. Unlike conventional retroviral vectors based on, for example, murine leukemia viruses (MLVs), lentiviral vectors, such as those derived from HIV and FIV, integrate into the genomes o f both dividing and nondividing cells.

Glaucoma is a particularly appropriate disease to investigate for corrective gene therapy because o f its chronicity and anatomically restricted pathology. However, this approach would require stable expression o f candidate genes in the aqueous outflow tract. Correcting the primary aqueous outflow pathophysiology, and the

complementary aim o f expressing antiapoptosis genes in retinal ganglion cells,^^^’

are both likely to require not only gene transfer to nondividing cells, but also transgene stability commensurate to the chronicity o f the disease. Trabecular meshwork cells are highly metabolically active and display pronounced phagocytic and secretory function, but appear to undergo limited cell division in vivo; less than 0.5% have been estimated to be mitotically active at any one time.^^^’ Although adenoviral and herpes simplex vectors transduce nondividing cells and can mediate gene transfer to the TM,^"^"^’ these vectors are best suited to short-term gene expression because they do not generate integrated transgenes. In addition, these vectors can trigger marked inflammatory responses. Oncoretroviral vectors, such as those derived from murine leukemia viruses (MuLYs), do achieve stable integration, but only in target cells that are proliferating at the time o f t r a n s d u c t i o n . T h e latter requirement could hinder effective gene transfer to the trabecular meshwork, although this question has not been investigated.

In contrast to these vectors, lentiviral vectors integrate permanently into the genomes o f both dividing and nondividing cells. This property, which enables the universal lentiviral strategy o f propagating through tissue macrophages, has been attributed to multiple determinants in virion proteins^"^^ and within the reversed transcribed DNA^"^^

that facilitate nuclear import o f lentiviral preintegration complexes. Envelope

Introduction: Gene Therapy

glycoprotein-pseudotyped lentiviral vectors have been derived from primate lentiviruses^"^^’ and from nonprimate animal lentiviruses/'^^^"^^^ Feline immunodeficiency virus (FIV) vectors exploit compartimentalized blocks to

productive cross-species infection, because they complete the postentry stages o f the infection cycle in nondividing human cells despite blocks to viral transcription and other life cycle mechanisms that prevent productive replication.^’ Human immunodeficiency virus type 1 (HIV-1) vector systems have so far received more extensive validation and molecular engineering for vector optimization.^^^

Additional features make glaucoma an intriguing potential proof-of-concept disease for gene therapy. The small amount o f tissue that would require targeting, and its accessibility, could enhance the feasibility o f adequate levels o f corrective gene transfer. In the absence o f a selective growth advantage for gene-altered cells, and/or a tissue that supports proliferation in vivo after ex vivo gene transfer,^^^ achieving permanent transduction o f most o f a relevant tissue has remained a major hurdle in most gene therapy situations. Specific tissue targeting is problematic if the target cells cannot be isolated ex vivo (e.g., ref.^^^). The anterior chamber can also be visualized through clinically feasible imaging methods, suggesting a means to monitor the in vivo expression o f an integrated transgene over time during gene therapy

developmental studies.

Long-term, stable, high-grade, and properly targeted transgene expression in the trabecular meshwork has not been achieved. Liposomes,^^^ adenovirus,^"^"^’ adeno- associated virus,^^^ and herpes simplex virus vectors^"^^ have been limited by short duration, inflammation, or lack o f sufficient, targeted transduction. In contrast to DNA virus- or plasmid-based vectors, lenti-retroviral vectors integrate permanently into the genomes o f transduced cells as an obligate part o f the life cycle.^^"^ The advantage over conventional onco-retroviral vectors is their ability to integrate in nondividing cells.

Not only could long-term transgene expression in the trabecular meshwork be utilized to study therapeutic transgenes, it would also enable transgenesis that is selective to the outflow tract and technically more simple than the generation o f entire transgenic animals. Prior to either therapeutic or experimental gene transfer, proof-of-principle has to be established with marker genes, the most common ones o f which are beta- galactosidase that allows intensely blue staining o f galactose breakdown products and

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-fluorescent proteins such as the humanized, enhanced green -fluorescent protein (eGFP), or a renilla reniformis green fluorescent protein (rGFP). The construction o f these vectors is described below in detail. LacZ as a marker gene has the advantage that it can be detected with the beta-galactosidase assay after short fixation and the blue product o f the assay is compatible with other conventional tissue staining techniques e.g. o f paraffin sections. It can be detected even at low multiplies o f infection (m.o.i.) or low enzyme activity. It can also be stained with

immunohistochemistry staining techniques as numerous antibodies are commercially available. A disadvantage is that it cannot be visualized in vivo easily. In contrast, eGFP and rGFP can be visualized in vivo with a variety o f techniques as long as the correct exciting wavelength is applied. E.g. standard cobalt blue light and observation with a slitlamp,^^ gonioscopic visualization with a histopathology microscope as described in the techniques section o f this thesis or excitation and capture with a scanning laser ophthalmoscope^^^ are possible.