AAV-Mediated Gene Therapy for Choroideremia: Preclinical Studies in Personalized Models

<div><p>Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1^st or 2^nd decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The <i>CHM</i> cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized <i>in vitro</i> models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the <i>CHM</i> cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.</p></div

Infection of wild type mouse retinas with AAV2.

<p><b>hCHM results in transduction of retinal cells and appears safe. I</b>). Western blot analysis of retinas of normal sighted control mice injected subretinally with 3E6 vg AAV2. hCHM shows one single expected size band in different animals (B, C) (i). A. Uninjected control. Immunolabeling of the AAV2. hCHM-injected retinas (I-ii) shows the localization of the REP-1 protein (Green) to photoreceptors and retinal pigment epithelium (I-ii-C). Nuclei are stained with DAPI. A- negative control, B-uninjected control retina. RPE: Retinal pigment epithelium; OS: Outer segments; IS: Inner segments; ONL: Outer nuclear layer; OPL: Outer plexiform layer; INL: Inner nuclear layer; IPL: Inner plexiform layer. <b>II).</b> TUNEL staining of CHO cells (II-i) and retinal sections (II-ii) from retinas injected with AAV2. hCHM in comparison to control uninjected tissue (II)<b>.</b> A: CHO cells were infected with E5 (II-i-C), and 2E5 (II-i-D) vg/ml of AAV2. hCHM to evaluate the cytotoxicity of the virus. There were few apoptotic nuclei 72 h after transduction, showing that infection did not result in acute cell death. There was no increase in TUNEL-positivity in AAV2. hCHM injected retinas (II-ii-D). Uninjected (II-ii-C) and injected retina appear similar (II-ii-D).Autofluorescence (red color) is observed in photoreceptor outer segments in all panels. Nuclei are stained with DAPI. Negative controls were generated by incubating the tissue with TUNEL reagents alone (i–A; ii–A). DNAse I treated cells or retinal sections were used as positive controls (i–B; ii–B).</p

Trafficking of RAB27 protein is restored in affected cells after infection with AAV2.

<p><b>hCHM.</b> CPF1 fibroblasts (i–vi) or CPS1 iPSCs (vii–xii) derived from CHM individuals showed improved trafficking of RAB27 after infection with AAV2. hCHM. In control CPF1 (i–iii) or CPS1 (vii–ix) untreated cells, Rab 27a (Green) was localized near the nucleus, whereas infection with AAV2. hCHM favored trafficking of RAB27 out of the perinuclear region in both CPF1(Rep-1 red, RAB27-green) (v–vi) and CPS1 (xi–xii) cells (REP1-green; RAB27-red). Nuclei are stained with DAPI and appear blue. <b>II).</b> Quantitative analysis of REP-1 and RAB27 levels in CHM iPSCs measured with imageStream. Histograms represent the increased level of exogenous REP-1 in cells infected with AAV2. hCHM (B) compared to controls (A) and unaffected wt controls (C). However, the level of REP-1 in transduced cells is reduced compared to unaffected control cells (E). Labeled Rab was increased in the surface mask in transduced cells (E) compared to uninfected cells (D). The levels of membrane-associated Rab 27 are comparable to the levels observed in unaffected wild type controls (F) Panel G and H shows representative cell images demonstrating the trafficking of Rab 27 to cell membrane in grey-scale. From left to right are shown: Brightfield, Rab 27, and REP-1, followed by composite images of REP-1 and RAB27. The cell surface masks used to define the inside and surface of the cell are overlayed in Brightfield and RAB/REP-1 labeled cells. White arrow in panel G, accumulated Rab inside the cell. Red arrowheads in panel H, presence of membrane Rab.</p

REP-1 protein is produced by fibroblasts [CPF1 (i, ii)] and iPSCs [CPF2 (iii, iv)] following AAV2.

<p><b>hCHM infection</b>: Fibroblasts and iPSCs of CHM individuals were infected with 2×10⁵ vg/cell AAV2. hCHM and production of REP-1 was assessed by immunofluorescence (i); western blot analysis (ii); and flow cytometry (iii, iv). Cytoplasmic distribution pattern of REP-1 was confirmed by co-staining with anti-actin antibody (i). Western blot analysis (ii) further confirmed the presence of REP-1 protein. FACS analysis of CHM iPSCs infected with AAV2. hCHM showed a high level of REP-1 protein compared to untransduced controls (iii,iv). Immunofluorescence showed high levels of REP1 protein in cells infected with AAV2. hCHM (vi) compared with controls (v). Nuclei are stained with DAPI. Scale bar is 50 uM. Data are representative of 3 independent experiments.</p

Prenylation activity in fibroblasts (CPF1) (i) and iPSCs (CPS1, CPS2) (ii) cultured from CHM individuals is restored following infection with AAV2.

<p><b>hCHM.</b> Prenylation assay was performed using the cytosolic fraction of cells transduced with AAV2. hCHM and from untreated affected cells (Control). Cell lysates were incubated with RabGGTase, RAB27 and [3H]-labeled GGPP. A significant increase (P<0.02) in the prenylation activity of exogenous REP-1 was observed in both CPF1 (2 fold) and CPS1, CPS2 cells (∼3 fold).</p

Generation and Characterization of AAV2. hCHM.

<p>I). Schematic of the AAV proviral plasmid carrying human <i>CHM</i>. under the control of the cytomegalovirus enhancer chicken beta actin (eCBA) promoter. ITR: Inverted terminal repeats; Ori: Replication origin; KanR: Kanamycin resistance gene. II) i) Immunoblot and ii) fluorescent analysis reveals REP-1 protein in CHO cells transfected with pAAV2.hCHM. Lane A: Transfected cell (25 ug protein), B: Control (untransfected) cells, C- protein marker. Immunocytochemical analysis revealed the localization of REP-1 to the cytosolic region (II-ii-B; Green). No REP1 is observed in control cells (II-ii-A). Nuclei are stained with DAPI and appear blue. Scale bar is 50 uM. <b>III)</b> Immunoblot analysis of CHO cells infected with 1E3-2E5 viral genomes (vg) of AAV2. hCHM show an increase in REP-1 protein (indicated by arrow) proportional to the titer. Positive (+ve) control: pAAV2. hCHM-transfected CHO cell lysate.</p