An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications

Abstract

The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl β-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.

Figure 1. Comparison of constitutively and autogenously regulated transgene systems in E. coli and HEK293T cells.

(A, B) Schematic diagram of the bacterial (A) classically regulated expression system (CRES) and (B) autogenously regulated expression system (ARES) tested in panels (C, D). Promoters, regulator genes, reporter genes, and operator sequence are indicated. (C) Mean normalized YFP fluorescence as a function of IPTG concentration for both the CRES and ARES in E. coli. Data were normalized to an E. coli tranformant expressing YFP under the control of a constitutive promoter. Data points represent mean +/− SEM, n = 5. (D) Mean normalized mCherry fluorescence of the lacI-mCherry fusion as a function of IPTG concentration for both CRES and ARES in E. coli. Data were normalized to an E. coli tranformant expressing lacI-mCherry under the control of a constitutive promoter. Data points represent mean +/− SEM, n = 5. (E, F) Schematic diagram of the eukaryotic (A) classically regulated expression system (CRES) and (B) autogenously regulated expression system (ARES) tested in panel (G). Promoters, regulator genes, reporter genes, polyadenylation sites, 2A cleavage signal, and operator sequence are indicated. (G) Mean YFP fluorescence as a function of IPTG concentration for both the CRES and ARES in transfected 293T cells. Data points represent mean +/− SEM, n = 3. (H) Mean luminescence as a function of IPTG concentration for the ARES encoding luciferase as a reporter in AAV-transduced 293T cells. Data points represent mean +/− SEM, n = 3.

Figure 2. The autogenous regulatory system is functional in mouse retina in vivo.

(A) Map of the autogenously regulated expression system (ARES) within an AAV production vector (AAV8.ARES.Luciferase). A CMV promoter controls the expression of both the lacI repressor and Luciferase, linked via a 2A peptide cleavage sequence. Orange boxes indicate lac operator sites. Intronic, polyadenylation, and AAV ITR sequences are indicated. (B) Live imaging of luciferase activity over a 33-day period in a representative animal subretinally injected with AAV8.ARES.luciferase in the right eye. (C) Live imaging of luciferase activity in the left, un-injected eye of the same animal as in panel (A). (D) Normalized integrated luminescence of the injected (right) eye were calculated by dividing the observed luminescence by the sum of luminescent measurements made in both the on and off states for each animal. Induction of luciferase in AAV injected eye increases significantly after administration of IPTG (P < 0.01). Green bars represent days of IPTG gavage. (E) The fold change was determined by evaluating the normalized integrated luminescence on day n relative to day m, where n and m are labeled on the x-axis as to illustrate dynamic regulation. A fold change >1 indicates induction of luciferase expression while fold change <1 indicates repression of luciferase expression. *p < 0.05, **p < 0.01, n = 8. (F) Histological sections of injected retinas from two representative animals stained with hematoxylin and eosin. (RPE, retinal pigmented epithelium, ONL, outer nuclear layer, INL, inner nuclear layer, GC, ganglion cell layer).