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AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models.

Vasireddy V, Mills JA, Gaddameedi R, Basner-Tschakarjan E, Kohnke M, Black AD, Alexandrov K, Zhou S, Maguire AM, Chung DC, Mac H, Sullivan L, Gadue P, Bennicelli JL, French DL, Bennett J - PLoS ONE (2013)

Bottom Line: 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 CHM 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.

View Article: PubMed Central - PubMed

Affiliation: F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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 CHM 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 in vitro 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 CHM 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.

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REP-1 protein is produced by fibroblasts [CPF1 (i, ii)] and iPSCs [CPF2 (iii, iv)] following AAV2.hCHM infection: 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.
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pone-0061396-g003: REP-1 protein is produced by fibroblasts [CPF1 (i, ii)] and iPSCs [CPF2 (iii, iv)] following AAV2.hCHM infection: 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.

Mentions: A line of fibroblasts corresponding to the iPSC line CPS1, named CPF1, was also generated as previously described. [30]Restoration of function mediated by AAV2. hCHM in vitro in CHM patient-derived cells: Patient-derived fibroblasts were infected with AAV2. hCHM at an MOI of 2×105 vg/cell and analyzed for the expression of REP-1 protein by immunofluorescence and western blot analysis. Representative results are shown for CPF1 (figure 3). Immunofluorescence analysis showed a lack of expression of REP-1 prior to exposure to AAV2. hCHM (data not shown). After infection, there was a predominant cytosolic localization of exogenous REP-1 (figure 3-i), as demonstrated with co-staining for actin. Immunoblot analysis of cells before and after infection with AAV2. hCHM demonstrated a lack of REP-1 in untreated cells whereas abundant REP-1 protein was present in treated cells (figure 3-ii, arrow).


AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models.

Vasireddy V, Mills JA, Gaddameedi R, Basner-Tschakarjan E, Kohnke M, Black AD, Alexandrov K, Zhou S, Maguire AM, Chung DC, Mac H, Sullivan L, Gadue P, Bennicelli JL, French DL, Bennett J - PLoS ONE (2013)

REP-1 protein is produced by fibroblasts [CPF1 (i, ii)] and iPSCs [CPF2 (iii, iv)] following AAV2.hCHM infection: 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646845&req=5

pone-0061396-g003: REP-1 protein is produced by fibroblasts [CPF1 (i, ii)] and iPSCs [CPF2 (iii, iv)] following AAV2.hCHM infection: 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.
Mentions: A line of fibroblasts corresponding to the iPSC line CPS1, named CPF1, was also generated as previously described. [30]Restoration of function mediated by AAV2. hCHM in vitro in CHM patient-derived cells: Patient-derived fibroblasts were infected with AAV2. hCHM at an MOI of 2×105 vg/cell and analyzed for the expression of REP-1 protein by immunofluorescence and western blot analysis. Representative results are shown for CPF1 (figure 3). Immunofluorescence analysis showed a lack of expression of REP-1 prior to exposure to AAV2. hCHM (data not shown). After infection, there was a predominant cytosolic localization of exogenous REP-1 (figure 3-i), as demonstrated with co-staining for actin. Immunoblot analysis of cells before and after infection with AAV2. hCHM demonstrated a lack of REP-1 in untreated cells whereas abundant REP-1 protein was present in treated cells (figure 3-ii, arrow).

Bottom Line: 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 CHM 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.

View Article: PubMed Central - PubMed

Affiliation: F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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 CHM 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 in vitro 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 CHM 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.

Show MeSH
Related in: MedlinePlus