Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy

Abstract

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.

Figure 1. Strategy for the GUCY2D^E837D/R838S transgenesis.

(A) Comparative analysis of the cone arrestin promoters from human, pig and mouse was performed. The 2 kbp upstream plus 200 bp downstream human and pig Arr3 promoters were aligned against that of the pig. Brown Conserved blocks with similarity higher than 70% were colored red under the curve. (B) The core promoter region was found to be highly conserved in pig, human and mouse. Four potential binding sites for the CRX photoreceptor transcription factor could be identified while the conserved TATA-boxes are located about −30 bp upstream of the published transcriptional start sites (TSS) , . TSS of the pig Arr3 gene was predicted by comparing the sequence with the JASPAR’s initiator weight matrix. Other promoter elements, such as CAAT-box, SP1, TATA-box associated with minor TSS, were not shown. (C) Schematic of the transgene cassette encoding the human mutant GUCY2D^E837D/R838S cDNA under the control of a fragment of the pig cone arrestin promoter. LTR: Long terminal repeat, the 3′ LTR is designated as “delta U3” for a fragment of the U3 region has been deleted, rendering the vector “self-inactivating”, i.e. unable to replicate once in the target cell, this also prevents interference of the HIV promoter with the internal Arr3 promoter; cPPT-CTS: central polypurine tract – Central termination site.

Figure 2. Expression of GUCY2D^E837D/R838S transcript in transgenic pigs.

Assessment of transgene expression by RT-PCR. Transgenic pigs: 904, 907, 908, 914, 915, 917, 918, 920, control non-transgenic pig (929). +: with reverse transcription; −: without reverse transcription; hGUCY2Dmut: GUCY2D^E837D/R838S PCR fragment; pig GUCY2D: pig GUCY2D PCR fragment; GAPDH: pig GAPDH PCR fragment.

Figure 3. Range of visual function in GUCY2D^E837D/R838S transgenic pigs.

(A) Traces of photopic electroretinogram recordings (ERG) at 11 weeks are shown for one representative control and several transgenic animals. (A, first column) single flash at 3 cds/m², (A, second column) single flash at 10 cds/m2, (A, third column) flickers at 3 cds/m²,30 Hz. (B) b-wave amplitudes for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (C) a-wave amplitudes obtained for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (D) Representation of the time needed to complete the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. (E) Representation of the errors (missing or striking into an obstacle), alternative prospections (the number of times individuals investigated an obstacle by sniffing or licking) and resulting scores from the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. Horizontal bars in B,C represent the mean of the different groups with the SEM; *: p<0.05;***: p<0.001; errors in E: miss or strike into an obstacles; alternative prospection in E: sniff or lick the obstacles; score in E: sum of errors and alternative prospections.

Figure 4. Abnormal retinal morphology in transgenic pigs.

(A) Morphological examination of a retina from a control animal reveals retinal layers: outer segments (OS), inner segment of photoreceptors (IS), photoreceptor nuclei (ONL). (B) Displaced nuclei were observed in the outer segment layer in transgenic retina (arrows). (C) Quantification of the number of displaced nuclei in transgenic and control animals. (D,E,F) Immunolabeling for specific cone markers PNA and M-opsin in transgenic retina identified most of these displaced cells as cones. (G,H) Immunolabeling for specific cone markers PNA and M-opsin in control retina. (I) Quantitation of relative density of displaced cones as determined by PNA or M-opsin labeling across 100 µm on the section. OS: outer segment; IS: inner segment; ONL: outernuclear layer; PNA: peanut agglutinin; M-opsin: medium wavelength opsin; nb: number. Scale bar in A, B and D to F represents 50 µm.

Figure 5. Case reports: pig 920.

Grouped data collected for the transgenic pig 920 (4 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 920 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–L) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), and GFAP (H,I) in the central region of the retina of pig 920 (E–H) and a non-transgenic control (I–L). Arrows in E, G, I and K show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–L represents 50 µm.

Figure 6. Case reports: pig 917.

Grouped data for the transgenic pig 917 (3 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 917 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–P) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), GFAP (H,I) and S-opsin (I,J,O,P) in the central region of the retina of pig 917 (E–J) and a non-transgenic control (K–P). Arrows in E, G, K and M show examples of M-opsin positive outersegment, arrowhead in G shows displaced nuclei, arrowhead in J shows a displaced nucleus in a S-opsin positive cell. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–J represents 50 µm.

Figure 7. Case report: pig 908.

Grouped data for the transgenic pig 908 (2 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 908 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). For technical reasons, the time to reach the end of the obstacle course has not been measured at 52 weeks. (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–G) Immunolabeling for M-opsin (E), PNA (F) and merged picture (G) in the central region of the retina of pig 908. Arrows in E and G show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–G represents 50 µm.