1. Development of a Minimal Feline Immunodeficiency Viral Vector System and Scaled-up Production

Glaucoma, a leading cause of irreversible blindness, is a lifelong, progressive optic neuropathy that is caused by increased intraocular pressure. No long-term treatment was available to match its chronicity while being specific to the diseased tissues. As a postdoctoral scientist, I established high-level gene transfer with our minimal FIV vector system to the strainer tissue of the outflow tract, the trabecular meshwork. I created minimal transfer vectors for a novel lentiviral vector system based on the feline immunodeficiency virus (FIV) and explored them in eye models. I reduced the 1353 nucleotide gag gene stepwise to only 311 nucleotides needed to preserve packaging. This diminished chances of recombination with the packaging plasmid that other team members minimized. I lead and took part in several in vivo projects that showed sustained transgene function and enabled the first pressure lowering gene therapy.

1. Loewen N, Fautsch MP, Peretz M, Bahler CK, Cameron JD, Johnson DH, Poeschla EM. Genetic modification of human trabecular meshwork with lentiviral vectors. Hum Gene Ther. 2001 Nov 20;12(17):2109–2119. PMID: 11747600
2. Loewen N, Bahler C, Teo W-L, Whitwam T, Peretz M, Xu R, Fautsch MP, Johnson DH, Poeschla EM. Preservation of aqueous outflow facility after second-generation FIV vector-mediated expression of marker genes in anterior segments of human eyes. Invest Ophthalmol Vis Sci. 2002 Dec;43(12):3686–3690. PMID: 12454037
3. Loewen N, Fautsch MP, Teo W-L, Bahler CK, Johnson DH, Poeschla EM. Long-term, targeted genetic modification of the aqueous humor outflow tract coupled with noninvasive imaging of gene expression in vivo. Invest Ophthalmol Vis Sci. 2004 Sep;45(9):3091–3098. PMID: 15326125
4. Loewen, R. T., Roy, P., Park, D. B., Jensen, A., Scott, G., Cohen-Karni, D., Fautsch, M.P., Schuman, J.S., Loewen, N. A. A Porcine Anterior Segment Perfusion and Transduction Model With Direct Visualization of the Trabecular Meshwork. Invest Ophthalmol Vis Sci. 2016 Mar;57(3), 1338–1344.

2. Outflow Engineering for Glaucoma

The trabecular meshwork, a strainer-like tissue at the intake of the outflow system, is a principal cause of increased intraocular pressure in glaucoma. My laboratory developed a conditional cytotoxic vector that ablates this structure resulting in a pressure drop. A repopulation activity and increase in cell density follow this ablation suggesting a reservoir of progenitor cells. The findings contribute to the growing evidence that ocular stem cell niches exist adjacent to or within the diseased structures. Cytoablation has allowed us to replace the trabecular meshwork by seeding it with transplanted cells. We created the first ex vivo model for a highly challenging microincisional glaucoma surgery, ab interno trabeculectomy, and developed quantitative canalograms to measure the enhancement with a high spatial resolution. We used this technique to establish outflow tract vessel transduction and progenitor seeding. We correlated our functional studies to morphometric analyses of whole specimen ribbon-scanning confocal microscopy. These massive datasets indicated that proximal outflow structures had greater diameters nasally while the venous plexus was larger in the inferior limbus.

1. Zhang Z, Dhaliwal AS, Tseng H, Kim JD, Schuman JS, Weinreb RN, Loewen NA. Outflow tract ablation using a conditionally cytotoxic feline immunodeficiency viral vector. Invest Ophthalmol Vis Sci. 2014 Feb;55(2):935–940. PMCID: PMC3929079
2. Loewen RT, Brown EN, Scott G, Parikh H, Schuman JS, Loewen NA. Quantification of Focal Outflow Enhancement using Differential Canalograms. Invest Ophthalmol Vis Sci. 2016 May 1;57(6):2831-8. doi: 10.1167/iovs.16-19541. PMID: 27227352
3. Parikh HA, Loewen RT, Roy P, Schuman JS, Lathrop KL, Loewen NA. Differential canalograms detect outflow changes from trabecular micro-bypass stents and ab interno trabeculectomy. Sci Rep. 2016 Nov 4;6:34705. doi: 10.1038/srep34705. PMID: 27811973
4. Dang Y, Waxman S, Wang C, Loewen RT, Sun M, Loewen NA. A porcine ex vivo model of pigmentary glaucoma. Sci Rep. 2018 Apr 3;8(1):5468. PMCID: PMC5882895
5. Waxman S, Loewen RT, Dang Y, Watkins SC, Watson AM, Loewen NA. High-Resolution, Three Dimensional Reconstruction of the Outflow Tract Demonstrates Segmental Differences in Cleared Eyes. Invest Ophthalmol Vis Sci. 2018 May 1;59(6):2371-2380. doi: 10.1167/iovs.17-23075. PMID: 29847643
6. Waxman S, Wang C, Dang Y, Hong Y, Esfandiari H, Shah P, Lathrop KL, Loewen RT, Loewen NA. Structure-Function Changes of the Porcine Distal Outflow Tract in Response to Nitric Oxide. Invest Ophthalmol Vis Sci. 2018 Oct 1;59(12):4886–4895. PMCID: PMC6181305
7. Chen S, Waxman S, Wang C, Atta S, Loewen R, Loewen NA. Dose-Dependent Rho-Kinase Inhibitor Effects on the Distal Outflow Tract. Graefes Arch Clin Exp Ophthalmol. 2020 Jun;258(6):1211-1216. doi: 10.1007/s00417-020-04691-y. Epub 2020 May 5.
8. Loewen RT, Waxman S, Wang C, Chen S, Watkins SC, Watson AM, Loewen NA. 3D-Reconstruction of the Human Conventional Outflow System by Ribbon Scanning Confocal Microscopy. PLoS One. 2020 May 18;15(5):e0232833. doi: 10.1371/journal.pone.0232833. eCollection 2020.
9. Waxman S, Wang C, Dang Y, Loewen RT, Loewen NA. Tissue-engineered anterior segment eye cultures display intraocular pressure maintenance. Preprint. July 2019. https://paperpile.com/shared/ybyKqo

3. Techniques and Outcomes of Microincisional Glaucoma Surgery

Progress in microengineering has allowed manufacturing surgical devices small enough to ablate the trabecular meshwork, the primary outflow resistance in glaucoma. However, despite removal over up to 180 degrees early and late failures are common even in experienced hands. Evidence points towards an overlooked resistance from outflow tract elements that are downstream of the trabecular meshwork and can regulate flow. We identified risk factors and expanded indications for ab interno trabeculectomy that have led to broader usage even in glaucomas with a relative contraindication, for instance, glaucomas with narrow angles. I was the principal investigator for these research projects that analyzed outcomes in more than 1000 patients.

1. Bussel II, Kaplowitz K, Schuman JS, Loewen NA, Group TS, Others. Outcomes of ab interno trabeculectomy with the trabectome after failed trabeculectomy. Br J Ophthalmol. 2014 Aug 28;99(2):258–262. PMID: 25170064
2. Bussel II, Kaplowitz K, Schuman JS, Loewen NA. Outcomes of ab interno trabeculectomy with the trabectome by degree of angle opening. Br J Ophthalmol. 2015 Jul;99(7):914–919. PMCID: PMC4501175
3. Parikh HA, Bussel II, Schuman JS, Brown EN, Loewen NA. Coarsened Exact Matching of Phaco-Trabectome to Trabectome in Phakic Patients: Lack of Additional Pressure Reduction from Phacoemulsification. PLoS One. 2016 Feb 19;11(2):e0149384. PMCID: PMC4760733
4. Loewen RT, Roy P, Parikh HA, Dang Y, Schuman JS, Loewen NA. Impact of a Glaucoma Severity Index on Results of Trabectome Surgery: Larger Pressure Reduction in More Severe Glaucoma. PLoS One. 2016 Mar 23;11(3):e0151926. PMCID: PMC4805208
5. Esfandiari, H., Shazly, T., Waxman, S., Kola, S., Kaplowitz, K. B., Brown, E. N., & Loewen, N. A. (2018). Similar Performance of Trabectome and Ahmed Glaucoma Devices in a Propensity Score Matched Comparison. J Glaucoma. 2018 Jun;27(6):490-495. doi: 10.1097/IJG.0000000000000960. PMID: 29613976
6. Esfandiari H, Shah P, Torkian P, Conner IP, Schuman JS, Hassanpour K, Loewen NA. Five-year clinical outcomes of combined phacoemulsification and trabectome surgery at a single glaucoma center. Graefes Arch Clin Exp Ophthalmol. 2018 Sep 26; Available from: http://dx.doi.org/10.1007/s00417-018-4146-y PMID: 30259089
7. Esfandiari H, Taubenslag K, Shah P, Goyal S, Weiner AJ, Severson ML, Weiner A, Grover DS, Bussel II, Loewen NA. Two-year data comparison of ab interno trabeculectomy and trabecular bypass stenting using exact matching. J Cataract Refract Surg 2019 45:608–614
8. Al Yousef Y, Strzalkowska A, Hillenkamp J, Rosentreter A, Loewen N. A. Comparison of a second-generation trabecular bypass (iStent inject) to ab interno trabeculectomy (Trabectome) by exact matching. medRxiv peprint. October 2019.