Re­search­ers

Scien­tific back­ground

In neovas­cu­lar, fast evolving nAMD, the in­growth of ves­sels in­to the reti­na (choroid­al neovas­cu­lar­isa­tion, CNV) af­fects the physiolo­gic­al in­ter­ac­tions between Bruch’s mem­brane, reti­nal pig­ment ep­ithe­lial (RPE) cells and photorecep­tors, lead­ing to cells’ de­gen­er­a­tion and vis­ion loss. An over­ex­pres­sion of the an­giogen­ic vas­cu­lar en­dotheli­al growth factor (VEGF) com­pared to a de­creased ex­pres­sion of the anti-an­giogen­ic pig­ment ep­ithe­li­um-de­rived factor (PEDF), is mainly re­spons­ible for the CNV of por­ous, leaky ves­sels and con­sec­ut­ive mac­u­lar hem­or­rhages and ed­ema.

The avas­cu­lar, slow evolving aAMD re­sults mainly from in­flam­ma­tion and oxi­dative stress, caus­ing cel­lu­lar dys­func­tion that pro­vokes ac­cu­mu­la­tion of de­pos­its and the de­crease of RPE-synthe­sized neuro­pro­tect­ive factors, and fi­nally, the loss of photorecep­tors and reti­nal gan­gli­on cells after RPE cells’ death.

RPE cells pro­duce and secrete PEDF, which pos­sesses neuro­pro­tect­ive and anti-an­giogen­ic prop­er­ties, in par­tic­u­lar by in­hib­it­ing VE­GF-me­di­ated prolife­ration and mi­gra­tion of en­dotheli­al cells, and by pro­tect­ing RPE cells and neur­ons from oxi­dative stress and ischemia. GM-CSF is pro­duced by di­verse types of cells in­clud­ing the RPE and is re­spons­ible for coun­ter­act­ing ap­op­tos­is, prolife­ration, dif­fer­en­ti­ation and mat­ur­a­tion of my­el­oid cells, and ad­apt­ive im­mune re­sponses to in­flam­ma­tion and in­fec­tion. PEDF alone is suf­fi­cient to ef­fect­ively in­hib­it CNV in nAMD. Over­ex­pressed toge­ther, PEDF and GM-CSF have the po­ten­tial to pre­vent reti­nal de­gen­er­a­tion in aAMD, by mit­ig­at­ing the ef­fects of anti-oxi­dative stress, in­hib­it­ing in­flam­ma­tion, sup­port­ing cell sur­viv­al, al­low­ing the in­teg­ra­tion of cell trans­plants and pro­tec­tion of neur­al cells. To al­le­vi­ate dam­ages caused by both forms of AMD and /or to ease treat­ment, cell-based gene the­ra­py might thus rep­res­ent a power­ful tool to over­ex­press these pro­teins ex­ert­ing a protec­tive ef­fect on the reti­na.

We pro­pose to trans­plant sub­ret­in­ally iris pig­ment ep­ithe­lial (IPE) or RPE cells after ge­net­ic modi­fic­a­tion, to en­able perma­nent over­ex­pres­sion of PEDF and/or GM-CSF. Trans­fec­tion is suc­cess­fully rea­lized us­ing the Sleep­ing Beauty (SB100x) trans­po­son sys­tem, that of­fers an ef­fi­cient and safe meth­od for gene de­liv­ery. We have shown that IPE and RPE cells can be ef­fi­ciently trans­fec­ted with the PEDF and the GM-CSF genes ex­pressed in vitro for longer than 18 months us­ing SB100X and con­firmed the cell protec­tive, anti-an­giogen­ic, ‑in­flam­mat­ory and ‑ox­id­ant ef­fect of PEDF/GM-CSF in vitro and reti­na cul­ture. We demon­strated that trans­planted PEDF-trans­fec­ted cells cause a sig­ni­fic­ant re­duc­tion of neovas­cu­lar le­sions in vivo.

Safety of the vec­tor sys­tem has been im­proved by us­ing anti­bio­tic-res­ist­ance-gene free mini­plas­mids. Ad­di­tion­ally, we es­tab­lished the trans­fec­tion us­ing the trans­posase mRNA in­stead of DNA, which is de­graded with­in ap­prox­im­ately 2 minutes. Thus, a long-term ex­pres­sion of the trans­posase that poses the risk of re-mo­bil­iz­a­tion and ‑in­teg­ra­tion of the trans­po­son with an in­creased risk of ad­verse ef­fects such as in­ser­tion­al muta­gen­es­is is min­im­ized.

Ac­cord­ing to the num­ber of cells we can col­lect from an iris sample for trans­fec­tion (about 10,000), our gene the­ra­py is suited to treat early stages of AMD be­fore cell loss.

For ad­vanced stages, when RPE cells are lost, we aim to com­bine our gene the­ra­py with stem cell ap­proaches.

Eth­ic­ally fa­vor­able, auto­log­ous iPS cells shall be re­pro­grammed from non-inva­sively col­lec­ted ren­al tu­bu­lar cells us­ing the SB100x sys­tem and dif­fer­en­ti­ated. The gen­er­ated RPE cells will be en­hanced to re­cov­er a healthy reti­nal en­vir­on­ment to en­sure the sur­viv­al of them­selves and reti­nal cells by afore­men­tioned gene the­ra­py ap­proaches.