Targeted CNS Delivery Using Human MiniPromoters and Demonstrated Compatibility with Adeno-Associated Viral Vectors

Abstract

Critical for human gene therapy is the availability of small promoter tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters using computational biology and phylogenetic conservation. MiniPromoters were tested in mouse as single-copy knock-ins at the Hprt locus on the X Chromosome, and evaluated for lacZ reporter expression in CNS and non-CNS tissue. Eighteen novel MiniPromoters driving expression in mouse brain were identified, two MiniPromoters for driving pan-neuronal expression, and 17 MiniPromoters for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPromoters exhibit similar cell-type specificity when delivered via adeno-associated virus (AAV) vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy number effects or genomic location, and results in constructs predisposed to success in AAV. These MiniPromoters are immediately applicable for pre-clinical studies towards gene therapy in humans, and are publicly available to facilitate basic and clinical research, and human gene therapy.

Keywords: AAV; gene therapy; promoters.

Figure 1

Brain expression patterns of 18 novel MiniPromoters and 2 additional MiniPromoter strains that were remade with the lacZ reporter. Adult mice were perfused, brains postfixed, sectioned sagittally or coronally at 1 mm, and stained overnight for lacZ using the substrate X-gal. At least two germline animals were analyzed for each established strain. Ple12 (AVP RRs) expressed in amygdala and thalamus (top panel), and in the ventral hippocampus and cortex layer IV (bottom panel). Ple15 (C8ORF46 RRs) expressed throughout the cortex and throughout the hippocampal pyramidal and dentate granule neurons with strong signal in the dentate gyrus. Ple16 (C8ORF46 RRs) showed a similar but weaker expression pattern to Ple15. Ple22 (CCKBR RRs) had lacZ-positive cells located in the periaqueductal gray (top panel, arrow) and in the hypothalamus (bottom panel, arrow). Ple23 displayed cortical staining throughout the brain, colliculi staining, a strong localized area at the anterior thalamus (top panel, arrow), and in the midbrain colliculi (bottom panel, arrow head), cerebellar granular and Purkinje cells (bottom panel, arrow). Ple27 and Ple28 (CCL27 RRs) showed expression throughout most brain regions, with Ple28 being much stronger. Ple29 (CCL27 RRs) expressed weakly in small localized areas of the thalamus and hypothalamus (bottom panel, arrow and arrowhead, respectively). Ple67 (FEV RRs) expressed in cortical layers II/III, cerebellar Purkinje cells, and strongly throughout most midbrain and hindbrain areas, with a definitive exclusion of striatal tissue (top panel). Raphe nuclei are clearly positive (bottom panel). Ple97 (GPX3 RRs) stained cells localized at the anterior thalamus (top panel, arrow) and ventrolateral hippocampus (bottom panel, arrow), and also in large blood vessels (top panel, arrow head). Ple112 (HCRT RRs) expressed in the anteroventral thalamus (top panel, arrow) and lateral hypothalamic area (top panel, arrow head). The lateral geniculate complex was positive (bottom panel, arrow). Ple122 (ICMT RRs) expressed strongly throughout all major brain regions, with comparatively weaker expression found in the thalamus, and hippocampal CA2, 3, 4 and dentate gyrus (top panel). Ple146 (NTSR1 RRs) had scattered staining in the anterior thalamus (top panel, arrow) and in Purkinje cells (bottom panel, arrow). Ple155 (PCP2 RRs) expressed strongly in the anterodorsal, anteroventral, and anteromedial nuclei (top panel, arrow). Additional staining was seen in the retrosplenial area (bottom panel, arrow). Ple170 (POGZ RRs) expressed strongly throughout all brain regions, with weaker staining in the striatal nuclei. The olfactory bulb mitral, glomerular, and granular layers are all stained (bottom panel). Ple201 (SLC6A5 RRs) expressed weakly in the anterior and retrosplenial areas of the cortex (top panel, arrow). Staining was present in the medulla also (bottom panel, arrows). Ple232 (TNNT1 RRs) expressed in the anterior cingulate and zona incerta (top panel, arrow and arrow head, respectively) and in the lateral hippocampus (bottom panel, arrow). Ple235 (TRH RRs) expressed in the pontine grey and periaqueductal grey (top and bottom panels respectively, arrows). Ple238 (TRH RRs) had scattered expression in many brain regions, including the cerebellar interposed and fastigial nuclei and in parts of the medulla (bottom panel). Ple240 (UGT8 RRs) showed strong fiber-tract associated staining in all parts of the brain, with cortex layer IV strongly positive and in thalamic intralaminar and anterior nuclei groups. In addition, the olfactory bulb glomeruli and corpus callosum were clearly labeled. Bs, brainstem; CA, cornus ammonis; Cb, cerebellum; CH, chimera; Ctx, cortex; Hipp, hippocampus; Hth, hypothalamus; Mb, midbrain; RRs, regulatory regions; Str, striatum; Th, thalamus.

Figure 2

Two MiniPromoters, Ple26 (CCL27 RRs) and Ple170 (POGZ RRs), drive near pan-neuronal expression throughout the mouse brain. For each, a representative 1-mm sagittal and coronal whole brain section is shown, as well as cryosections of NeuN (neuronal marker) and Gfap (glial marker) antibody co-labeling with X-gal in the cortex, hippocampus, and cerebellum. (a) Ple26 (CCL27 RRs) showed marked expression throughout the adult brain with slightly less labeling in the olfactory bulb. In the cortex, NeuN closely co-labeled many of the X-gal positive cells (black arrows). However, there were some NeuN cells that do not co-label with X-gal (red arrows), as well as many smaller punctate X-gal nuclei that do not co-label with NeuN (white arrows). Nearly all hippocampal neurons co-label with X-gal, and extensive co-labeling was also observed in the cerebellum, with X-gal only staining also present in the molecular layer. Also, rare X-gal/Gfap-positive cells were observed (black arrows). (b) Ple170 (POGZ RRs) demonstrated a similar expression pattern to that of Ple26. The CA2 and CA3 regions of the hippocampus showed less staining than the dentate gyrus and CA1 regions. Many NeuN co-labeled cells were visible (black arrows). However, some NeuN-positive cells in the cortex were not labeled with X-gal (red arrows), as well as some punctate X-gal nuclei were NeuN-negative (white arrows). Reduced NeuN co-labeling was seen in the hippocampus and cerebellum, particularly in the cerebellar granule cell layer. Occasionally, a cell was found to be double labeled with Gfap and X-gal (black arrows). CA, cornus ammonis; Cb, cerebellum; Ctx, cortex; Hipp, hippocampus; RRs, regulatory regions. (Scale bars = 100 μm).

Figure 3

Thirteen MiniPromoters expressed in the mouse spinal cord. LacZ expression was detected by X-gal immunohistochemistry in whole mount thoracic spinal cord. (a) Ple17 (C8ORF46 RRs), Ple28 (CCL27 RRs), Ple32 (CLDN5 RRs), and Ple170 (POGZ RRs) showed scattered expression in the central spinal cord and in nerve bundles. Ple24 (CCKBR RRs) expression was seen primarily in the spinal cord with rare puncta in the dorsal root ganglia (data not shown). Ple26 (CCL27 RRs), Ple53 (DCX RRs), and Ple55 (DCX RRs) expressed throughout the gray matter regions and also in dorsal root ganglia; the latter particularly strong for Ple53 and Ple55. Ple34 (CLDN5 RRs) and Ple123 (ICMT RRs) expressed throughout all of the spinal cord, while Ple123 demonstrated weaker expression in nerve bundles but strong expression in dorsal root ganglia. Ple88 (GFAP RRs) was expressed only in patches along white matter regions of the central spinal cord. Ple167 (POGZ RRs) expressed in patches in the spinal cord with some nerve fibers and occasional dorsal root ganglia puncta also positive (data not shown). Ple240 (UGT8 RRs) is expressed strongly and predominantly along spinal nerve cord bundles with weaker expression in the central spinal cord tissue and in dorsal root ganglia. (b) Summary of expression pattern for each construct in the three key regions of the spinal cord. CH, chimera; DRG, dorsal root ganglion; ND, not done; RRs, regulatory regions; √, expression present; ×, expression absent.

Figure 4

Expression of six MiniPromoters during development. E12.5 embryos were harvested and stained for lacZ expression using X-gal. Whole-mount (first panels) and partially cleared whole-mount embryos (second panels) were photographed. (a) Ple26 (CCL27 RRs) stained throughout the entire embryo. (b) Ple123 (ICMT RRs) displayed variability, with the most common expression pattern being limited to the developing heart as shown. (c) Ple131 (MKI67 RRs) was expressed throughout the embryo at varying levels, most prominently in the heart. (d) Ple140 (NR2E1 RRs) showed a very strongly defined pattern in the hypothalamus of the embryo. (e) Ple170 (POGZ RRs) demonstrated staining in all parts of the embryo, but generally stronger outside of the nervous system. (f) Ple232 (TNNT1 RRs) stained strongly in skeletal muscle and the tongue of the embryo but was not visible in the brain or spinal cord. RRs, regulatory regions.

Figure 5

Comparison of two DCX-based MiniPromoter constructs with similar but nonidentical expression patterns identifies putative functional TFBS. (a) Diagram showing the differences between Ple53 and Ple55. Both MiniPromoters contained a minimal “Prom” sequence of 2,353 bp; however, Ple53 contained an additional but contiguous sequence and thus was named “LongProm”, whereas Ple55 contained a noncontiguous segment 3 in addition to the Prom. (b) In the UCSC genome browser, predicted TFBS for Mafk and BRACH protein families were observed in the extended LongProm sequence specific to Ple53. Predicted RXR/RAR heterodimer and SRF sites were found in the segment 3 specific to Ple55. Comparison of the expression of lacZ using the substrate X-gal (blue) demonstrated that (c, e, g) Ple53-lacZ was expressed as a subset of the more expansive (d, f, h) Ple55-lacZ expression pattern. (c, d) The comparison demonstrated that with the Ple53 construct, the majority of cells labeled in the cortex were located in the deeper cortical layers, whereas with Ple55, they were distributed throughout the cortex. There were also more cells in Ple55 that did not co-label with NeuN. (e, f) Expression analysis in E12.5 X-gal stained whole-mount and partially cleared embryos for each MiniPromoter demonstrated that Ple55 contained all of the neuronal based expression observed in Ple53 plus staining in the precartilage or premuscle of the face and limb buds. Both MiniPromoters exhibited expression at the distal edge of the limb buds, and both included staining of the developing kidneys (white arrows). A close-up of the face and front limb showed the additional staining in Ple55 (black arrows). (g, h) (upper panels) MiniPromoter lacZ expression as detected by X-gal reaction in the retina (left panel). Expression for the DCX-based designs was limited to the ganglion cell layer (GCL) for both Ple53 and Ple55, with rare cells observed in the inner nuclear layer. NeuN (marking all retinal ganglion and amacrine cells) co-labeled with X-gal (center panel), but Gfap (retinal astrocytes) did not (right panel). Rare cells did not co-label with NeuN (white arrows). (g, h) (lower panels) Immunofluorescence co-labeling with Brn3, βtubIII, and Sox2. Co-labeling demonstrated that most lacZ-positive cells stained with βtubIII and Brn3 markers (ganglion cells) for both MiniPromoters (left and center panels), and only rarely with Sox2-positive cholinergic amacrine cells (right panel, white arrows). Ctx, cortex; Hipp, hippocampus; NR, neutral red; RAR, retinoic acid receptor; RRs, regulatory regions; RXR, retinoid X receptor; SRF, serum response factor; TFBS, transcription factor binding sites; UCSC, University of California, Santa Cruz. (Scale bars = 100 μm, except for (g, bottom panel) fluorescence images 20 μm).

Figure 6

Fifteen MiniPromoters expressed in the mouse eye. Mice were perfused, eyes harvested, and stained for lacZ using the substrate X-gal (blue). Eyes were cryoprotected, embedded, and sectioned. Ple24 (CCKBR RRs), Ple25 (CCKBR RRs), Ple32 (CLDN5 RRs), Ple67 (FEV RRs), and Ple146 (NTSR1 RRs) expressed in a subset of GCL cells. Rarely, an amacrine cell in the innermost part of the INL was positive in Ple24, or a rare positive INL cell in Ple32. Ple26 and Ple28 (both based on CCL27 RRs) expressed in the INL, and in photoreceptors as evidenced by the columnar staining of the ONL and photoreceptor segments. Ple26 showed extensive IPL and OPL fiber staining and Ple28 was also positive in the GCL. Ple34 (CLDN5 RRs) and Ple155 (PCP2 RRs) have staining characteristic of bipolar cells, with cell bodies in the INL and processes extending to the GCL (Ple34) or inner half of the IPL (Ple155). Ple88 (GFAP RRs) stained in the retinal astrocytic layer. Ple123 (ICMT RRs) expressed throughout the retina. Ple131 (MKI67 RRs) expressed in cells of the corneal epithelium. Ple167 and Ple170 (both based on POGZ RRs) demonstrated similar staining patterns in the GCL, INL, OPL and photoreceptor segments. Ple240 (UGT8 RRs) had fiber-like staining spanning the IPL and clear cell body labeling in a subset of INL cells. CH, chimera; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RRs, regulatory regions. (Scale bar = 100 μm).

Figure 7

Three MiniPromoters that expressed in the retinal ganglion cell layer in knock-in mice maintained this restricted expression from AAV viral vectors. (a) Transduction profile of the AAV2(quad Y-F) vector with the CBA promoter driving GFP. Three MiniPromoter constructs (b, c) Ple25, (d, e) Ple53, and (f, g) Ple67, which expressed in the GCL when docked 5′ of Hprt driving β-galactosidase, were recloned into AAV2(quad Y-F) driving hGFP. (b, d, f) Knock-in mouse retinas stained for lacZ using the substrate X-gal (blue) and counterstained with neutral red (first panel) showed similar ganglion expression to intravitreal injected AAV2(quad) with the MiniPromoter driving hGFP (second panel, epifluorescence). (c, e, g) Virus-injected retinas were immunostained for Brn3a (a marker of ganglion cells). First panel shows Brn3a (red) with DAPI (blue), second panel hGFP (green) with DAPI (blue), and third panel the merge (yellow) of Brn3a (red), hGFP (green), with DAPI (gray). CBA, chicken beta-actin promoter; DAPI, 4′,6-diamidino-2-phenylindole; GCL, ganglion cell layer; GFP, green fluorescent protein; INL, inner nuclear layer; ONL, outer nuclear layer; RRs, regulatory regions. (Scale bars = 100 μm, except for fluorescence images 20 μm).