A survey of cytokine production by cultured human melanoma cells and characterization of melanoma-derived interleukin-1

This thesis examines the hypothesis that melanoma cells produce immunomodulatory and growth regulatory cytokines which influence tumor growth and progression. Screening assays of melanoma conditioned media demonstrated bioactivity for IL-1, TGF-$\beta$, and IFN, but not IL-2. Immunoassays detected IL-1$\alpha$, IL-1$\beta$, IL-6, IL-8, and GM-CSF, but not IL-3, IL-4, TNF-$\alpha$, or IFN-$\gamma$. Melanoma-derived IL-1 was characterized: Constitutive secretion and/or cell surface expression occurred in more than 50% of cultures, with poly(I):poly(C) causing upregulation and r-IL-1$\alpha$ causing down-regulation. Melanomas secreting IL-1 expressed 2.2 and 1.6 kb transcripts hybridizing IL-1$\alpha$ and IL-1$\beta$ probes, respectively. When cloned and sequenced from cell line WM1158, the melanoma IL-1$\beta$ gene was identical to the monocyte gene; but several clones of the IL-1$\alpha$ gene contained two variations of unknown significance. Although melanoma and monocyte IL-1 are products of the same genes, differences were found in their expression. LPS-treated monocytes secreted at least 10-fold more IL-1 than cultured melanomas, in a ratio of 1 (IL-1$\alpha$) to 10 (IL-1$\beta$); while IL-1$\alpha$ predominated in melanomas. While cell-associated IL-1 precursors were identical in size, secreted melanoma IL-1 was larger than monocyte IL-1. In addition, LPS, silica, and zymosan failed to induce IL-1 secretion in a panel of melanoma cell lines, suggesting that in contrast to monocytes, melanoma IL-1 expression is not a generally inducible phenomenon. Finally, evidence is presented for an IL-1 autocrine growth-regulatory loop in some melanomas. Cultured melanomas expressed mRNA for IL-1 type I and II receptors, and exogenous IL-1$\alpha$ altered proliferation in 3/4 lines which expressed receptor message. The effects of exogenous IL-1 were influenced by culture conditions. Under suboptimal conditions, IL-1 caused either an acceleration (1/4 lines) or delay (1/4 lines) in the onset of quiescence, and culture with MAb to r-IL-1$\alpha$ resulted in the opposite effects. In W489PF medium, cultures underwent sustained proliferation, and IL-1$\alpha$ either decreased (2/6 lines) or had no effect (4/6 lines) on (\sp3H) -thymidine incorporation. In conclusion, these data demonstrate that melanoma cells produce immunomodulatory and growth regulatory cytokines and suggest that both exogenous and endogenous IL-1$\alpha$ regulate the proliferation of some melanomas

Induced pluripotent stem cells as a model To test gene augmentation therapy for Choroideremia

Choroideremia (CHM) is an X-linked, blinding, inherited retinal degeneration that is symptomatic in childhood and leads to total blindness in midlife. The disease is characterized by the degeneration of photoreceptors, retinal pigment epithelium (RPE) and choriocapillaris. The causative factor for CHM was identifi ed as a defect in the Rab Escort Protein 1 (REP1) which is involved in prenylation of Rab proteins. The nature and onset of the disease and the size of the gene makes CHM an excellent target for recombinant adeno-associated virus (rAAV) mediated gene therapy. However, the animal model of this disease is not an exact replica of the condition and is not readily available. Therefore, we generated and evaluated induced pluripotent stems cells (iPSCs) from CHM subjects as in vitro models to explore the potential of gene augmentation therapy. A recombinant adeno-associated virus (AAV2) was generated that delivers the wild-type human CHM cDNA driven by a constitutive promoter. Infection of iPSCs with AAV2-CHM resulted in the expression of the CHM protein and rescue of enzymatic function in the defective cells. The gene transfer is effi cient and appears to be safe in the short-term, as shown by studies in affected cells and in normalsighted animal models. These studies not only aid in deciphering the pathogenetic mechanisms of the disease, but also pave the way for a human gene therapy clinical trial for CHM

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

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

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

Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial

Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study

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